Substituted γ-phenyl-Δ-lactams and uses related thereto

ABSTRACT

γ-Phenyl-substituted Δ-lactams are disclosed. They may be formulated into pharmaceutical compositions, and/or used in the treatment or prevention of inflammation or other conditions or disease states.

TECHNICAL FIELD

This invention is directed towards Δ-lactam compounds, and in particularto γ-Phenyl-substituted Δ-lactams, and therapeutic uses related thereto.

BACKGROUND OF THE INVENTION The Inflammatory Response (Inflammation)

Inflammation is an essential localized host response to invadingmicroorganisms or tissue injury which involves cells of the immunesystem. The classic signs of inflammation include redness (erythema),swelling (edema), pain and increased heat production (pyrema) at thesite of injury. The inflammatory response allows the body tospecifically recognize and eliminate an invading organism and/or repairtissue injury. Many of the acute changes at the site of inflammation areeither directly or indirectly attributable to the massive influx ofleukocytes (e.g., neutrophils, eosinophils, lymphocytes, monocytes)which is intrinsic to this response. Leukocytic infiltration andaccumulation in tissue results in their activation and subsequentrelease of inflammatory mediators such as LTB₄, prostaglandins, TNF-α,IL-1β, IL-8, IL-5, IL-6, histamine, proteases and reactive oxygenspecies for example.

Normal inflammation is a highly regulated process that is tightlycontrolled at several levels for each of the cell types involved in theresponse. For example, expression of the pro-inflammatory cytokine TNF-αis controlled at the level of gene expression, translation,post-translational modification and release of the mature form from thecell membrane. Many of the proteins up-regulated during inflammation arecontrolled by the transcription factor, NF-κB. Pro-inflammatoryresponses are normally countered by endogenous anti-inflammatorymechanisms such as generation of IL-10 or IL-4. A characteristic of anormal inflammatory response is that it is temporary in nature and isfollowed by a resolution phase which brings the state of the tissue backto its prior condition. The resolution phase is thought to involveup-regulation of anti-inflammatory mechanisms, such as IL-10, as well asdown-regulation of the pro-inflammatory processes.

Inflammatory Disease

Inflammatory disease occurs when an inflammatory response is initiatedthat is inappropriate and/or does not resolve in the normal manner butrather persists and results in a chronic inflammatory state.Inflammatory disease may be systemic (e.g. lupus) or localized toparticular tissues or organs and exerts an enormous personal andeconomic burden on society. Examples of some of the most common andproblematic inflammatory diseases are rheumatoid arthritis, inflammatorybowel disease, psoriasis, asthma, emphysema, colitis andischemia-reperfusion injury.

A common underlying theme in inflammatory disease is a perturbation ofthe cellular immune response that results in recognition of hostproteins (antigens) as foreign. Thus the inflammatory response becomesmisdirected at host tissues with effector cells targeting specificorgans or tissues often resulting in irreversible damage. Theself-recognition aspect of auto-immune disease is often reflected by theclonal expansion of T-cell subsets characterized by a particular T-cellreceptor (TCR) subtype in the disease state. Often inflammatory diseaseis also characterized by an imbalance in the levels of T-helper (Th)subsets (i.e., Th1 cells vs. Th2 cells).

Therapeutic strategies aimed at curing inflammatory diseases usuallyfall into one of two categories: (a) down-modulation of processes thatare up-regulated in the disease state or (b) up-regulation ofanti-inflammatory pathways in the affected cells or tissues. Mostregimes currently employed in the clinic fall into the first category.Some examples of which are corticosteroids and non-steroidalanti-inflammatory drugs (NSAIDs).

Many of the tissue, cellular and biochemical processes which areperturbed in inflammatory disease have been elucidated and this hasallowed the development of experimental models or assays to mimic thedisease state. These in-vitro assays enable selection and screening ofcompounds with a high probability of therapeutic efficacy in therelevant inflammatory disease. Thus, currently employed assays used tomodel the importance of the activated leukocytes in the development ofacute inflammation and maintenance of the chronic inflammatory state areassays monitoring leukocyte chemotaxis and cellular degranulation andcytokine synthesis and reactive oxygen species (ROS) production assaysin vitro. Since a result of acute or chronic neutrophil activation isrelease of ROS with resultant tissue damage, an assay for scavengers ofROS allows detection of compounds with potential therapeutic efficacy.Cellular assays to detect inhibitors of TNF-α release from stimulatedmacrophage or monocytic cells are an important component of an in vitromodel for inflammation as this cytokine is upregulated and has beenshown to contribute to the pathology in many inflammatory diseases.Since elevated cAMP in affected cells has been shown to modulate ordampen the inflammatory response, monitoring cellular cyclic AMP (cAMP)levels, and the activity of pathways controlling cAMP levels allows forthe detection of potential anti-inflammatory compounds. Assays mayinclude monitoring the level of cAMP itself, phosphodiesterase activity,or changes in cAMP response element (CRE)-luciferase activity.

Rheumatoid Arthritis

Rheumatoid arthritis (RA), the most common form of inflammatoryarthritis, is an auto-immune disorder of unknown etiology which affects1% of the adult population and is characterized by symmetric, chronic,erosive synovitis (inflammation of the joint synovial lining) andfrequent multisystem involvement. Interestingly, it is 3-6 times moreprevalent in women than men. Most patients exhibit a chronic fluctuatingcourse of disease that, if left untreated, results in progressive jointdestruction, deformity, disability, and premature death. Symptomsindicative of RA include pain and swelling of the joints (usuallysymmetrical), morning stiffness of joints and muscles, generalweakness/fatigue and fever and weight loss. RA results in more than 9million physician visits and more than 250,000 hospitalizations per yearin the U.S. each year. It frequently affects patients in their mostproductive years, and thus, disability results in major economic loss.

Recent insights have established that the genetic background, especiallythe structure of the class II major histocompatibilty (MHC) genes, playsa critical role in an individual's susceptibility and the severity ofthe disease. The current understanding of cytokine networks, chemokines,growth factors and adhesion molecules have led to the appreciation thatT cell-dependent and T cell-independent pathways contribute to theinitiation and perpetuation of rheumatoid arthritis. Furthermore, muchhas been learned about the specific cellular and biochemical eventsresponsible for the bone and cartilage destruction that characterizesthis disorder. At the tissue level, RA is characterized by synovialhyperplasia, hypertrophy, angiogenesis and attachment and invasion ofsynovial fibroblasts into adjacent cartilage and bone. In active RAthere are increased levels of the pro-inflammatory cytokines TNF-α, IL-1and IL-6 relative to the anti-inflammatory cytokines in affected joints.

Current Treatments for Rheumatoid Arthritis and Other InflammatoryDiseases

At present there is no cure or prevention (prophylactic) available forrheumatoid arthritis, only regimes that address symptoms such as painand stiffness. The five major treatment modalities for this diseaseinclude medication (pharmacological), physical (exercise), jointprotection and lifestyle changes and surgery.

Therapeutics for rheumatoid arthritis can be divided into three groups:nonsteroidal anti-inflammatory drugs (NSAIDs), disease modifyinganti-rheumatic drugs (DMARDs) also known as second line agents andcorticosteroids.

NSAIDs reduce pain at low doses and relieve some of the inflammatorysymptoms (swelling and stiffness) at higher doses through inhibition ofprostaglandin synthesis. Examples of non-prescription NSAIDs includeacetylsalicylic acid (ASA®, Aspirin®, Anacin®, etc.) and ibuprofen(Motrin®, Advil®, etc.). Examples of NSAIDs requiring a prescriptioninclude Naprosyn®, Relafen®, Indocid®, Voltaren®, Feldene® andClinoril®. Although these medications effectively address the acuteinflammatory component of rheumatoid arthritis, they only treat thesymptoms of and do not change the progression of the underlying disease.The deleterious side effects of NSAIDs can be serious with prolongedadministration and are mainly gastrointestinal (heartburn, bleeding orulcers).

DMARDs are often prescribed if inflammation persists for more than 6weeks or when the arthritis affects many joints simultaneously. They areusually administered in addition to a NSAID or steroid. Many DMARDs workby suppressing immune cells involved in the inflammatory response thusslowing progression of the disease. However, they are unable to reversepermanent joint damage. The most common drugs of this class are goldsalts, methotrexate, azathioprine, sulphasalazine, hydroxychloroquine,penicillamine and chloroquine. DMARDs often take several weeks forbeneficial effects to be seen and in many cases the exact mode ofefficacy in rheumatoid arthritis is unknown. Side effects are numerousincluding mouth sores, rashes, diarrhea and nausea. More serious sideeffects which necessitate careful monitoring through regular blood andurine tests include liver and kidney damage, excessive lowering of thewhite blood cell count (immune suppression) and platelet count (bloodclotting).

Corticosteroids are frequently prescribed in RA patients with extremeinflammation accompanied by severe pain, swelling and stiffness in thejoints. They are also used to treat systemic rheumatoid arthritis whichcan affect the lining of the lungs and blood vessels. The route ofadministration is usually oral (i.e., prednisone) but the drug can alsobe injected directly into the affected joint, vein, muscle oralternative site of inflammation. Side effects from long-term use ofsteroids in rheumatoid arthritis are serious and include cataracts, highblood pressure, muscle wasting, bruising, thinning of skin and bones,weight gain, diabetes and susceptibility to infection.

Even though only 5% of patients diagnosed with rheumatoid arthritis willgo on to develop more severe disease (involving debilitating andirreversible joint damage) those that do certainly do not have an idealset of therapeutics available to satisfactorily manage and/or cure thedisease. The currently available NSAIDs (even selective COX-2inhibitors) can successfully ameliorate the acute symptoms of rheumatoidarthritis such as swelling, pain and joint stiffness. However they donot affect either progression of joint destruction or effect anyreversal of articular or bone erosion. Second line drugs such as DMARD'sor corticosteroids may temporarily slow progression of the disease andreduce symptoms, but usually suffer from an unacceptable side-effectprofile or variable patient response and cannot reverse existing jointdamage. There is a significant need for therapeutic agents thateffectively arrest or reverse disease progression in rheumatoidarthritis.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a composition comprising acompound according to formula (1) and salts, solvates, isolatedstereoisomers, and mixtures thereof, and a pharmaceutically acceptablecarrier, diluent, or excipient,

wherein each of hydrogens H_(a), H_(b), H_(c), H_(d), H_(e), H_(f) andH_(g) may independently be replaced with a group selected from —W and—R⁷(W)_(n), and M₁ represents —W or —R⁷(W)_(n), wherein

W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸,—OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸, —PR⁸R⁸, —POR⁸,—PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸, —SONR⁸R⁸, —SO₂NH₂,—SO₂NHR⁸ and —SO₂NR⁸R⁸;

R⁷ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group whereinn of the hydrogen or halogen atoms of R⁷ are substituted by an equalnumber of W groups independently selected at each location;

R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group;

n is selected from 0, 1, 2, 3, 4 and 5; and

X is selected from —Br, —Cl, —F, —I.

In other aspects of the composition comprising a compound of formula(1): H_(a) and H_(b) are hydrogen; H_(a) is hydrogen and H_(b) is —W;H_(a) is hydrogen, H_(b) is —W and the carbon to which H_(b) is boundhas an S configuration; H_(a) is hydrogen, H_(b) is —W, and the carbonto which H_(b) is bound has an R configuration; H_(a) is hydrogen andH_(b) is —R⁷(W)_(n); H_(a) is hydrogen, H_(b) is —R⁷(W)_(n), and thecarbon to which H_(b) is bound has an S configuration; H_(a) ishydrogen, H_(b) is —R⁷(W)_(n), and the carbon to which H_(b) is boundhas an R configuration; H_(a) is hydrogen, H_(b) is —R⁷(W)_(n), H_(b) is—CH₂-phenyl, and phenyl has 0, 1 or 2 W substitutions; H_(c) is W; H_(d)and H_(e) are both hydrogen; H_(f) is W; H_(f) is selected from —OH and—OR⁸; H_(f) is selected from methoxy, ethoxy, propoxy, cyclopentyloxy,cyclohexyloxy, and benzyloxy; H_(f) is selected from —NH₂, —NHR⁸, and—NR⁸R⁸; H_(g) is —R⁷(W)_(n); M₁ is —W; M₁ is selected from methoxy,ethoxy, propoxy, cyclopentyloxy, cyclohexyloxy, and benzyloxy; M₁ isselected from —NH₂, —NHR⁸, and —NR⁸R⁸; M₁ is selected from —OH and —OR⁸;and/or M₁ is —R⁷(W)_(n). In one embodiment, none of H_(a), H_(b), H_(c),H_(d), H_(e), H_(f) or H_(g) is a heterocyclic ring.

The compound of formula (1) may have the stereochemistry of formula (1a)

The compound of formula (1) may have the stereochemistry of formula (1b)

A reference herein to compounds of formula (1) includes compounds offormulae (1a) and (1b).

In another aspect, the present invention provides a compositioncomprising a compound according to formula (2) and salts, solvates,isolated stereoisomers, and mixtures thereof, and a pharmaceuticallyacceptable carrier, diluent, or excipient,

wherein each of hydrogens H_(a), H_(b), H_(c), H_(d), H_(e), H_(f) andH_(g) may independently be replaced with a group selected from —W and—R⁷(W)_(n), and M₂ represents —W, wherein

W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸,—OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸, —PR⁸R⁸, —POR⁸,—PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸, —SONR⁸R⁸, —SO₂NH₂,—SO₂NHR⁸ and —SO₂NR⁸R⁸;

R⁷ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group whereinn of the hydrogen or halogen atoms of R⁷ are substituted by an equalnumber of W groups independently selected at each location;

R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group;

n is selected from 0, 1, 2, 3, 4 and 5; and

X is selected from —Br, —Cl, —F, —I.

In other aspects of compositions comprising a compound of formula (2):H_(a) and H_(b) are hydrogen; H_(a) is hydrogen and H_(b) is —W; H_(a)is hydrogen, H_(b) is —W, and the carbon to which H_(b) is bound has anS configuration; H_(a) is hydrogen, H_(b) is —W, and the carbon to whichH_(b) is bound has an R configuration; H_(a) is hydrogen and H_(b) is—R⁷(W)_(n); H_(a) is hydrogen, H_(b) is —R⁷(W)_(n), and the carbon towhich H_(b) is bound has an S configuration; H_(a) is hydrogen, H_(b) is—R⁷(W)_(n), and the carbon to which H_(b) is bound has an rconfiguration; H_(a) is hydrogen, and H_(b) is —CH₂-phenyl, where phenylhas 0, 1 or 2 W substitutions; H_(c) is W; H_(d) and H_(e) are bothhydrogen; H_(f) is hydrogen and H_(g) is W; H_(g) is selected from —OHand —OR⁸; H_(g) is selected from methoxy, ethoxy, propoxy,cyclopentyloxy, cyclohexyloxy, and benzyloxy; H_(g) is selected from—NH₂, —NHR⁸, and —NR⁸R⁸; H_(f) is hydrogen and H_(g) is —R⁷(W)_(n); M₂is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸, and—OR⁸; and/or M₂ is selected from —NH₂, —NHR⁸, —NR⁸R⁸, —OH, and —OR⁸. Ina preferred embodiment, none of H_(a), H_(b), H_(c), H_(d), H_(e), H_(f)or H_(g) may be replaced with a heterocyclic ring system.

The compound of formula (2) may have the stereochemistry of formula (2a)

The compound of formula (2) may have the stereochemistry of formula (2b)

A reference herein to compounds of formula (2) includes reference tocompounds of formulae (2a) and (2b).

In another aspect, the present invention provides a compound accordingto formula (3) and salts, solvates, isolated stereoisomers, and mixturesthereof,

wherein each of hydrogens H_(a), H_(c), H_(d), H_(e), H_(f) and H_(g)may independently be replaced with a group selected from —W and—R⁷(W)_(n), and H_(b) may be replaced with —W, wherein

W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸,—OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸, —PR⁸R⁸, —POR⁸,—PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸, —SONR⁸R⁸, —SO₂NH₂,—SO₂NHR⁸ and —SO₂NR⁸R⁸;

R⁷ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group whereinn of the hydrogen or halogen atoms of R⁷ are substituted by an equalnumber of W groups independently selected at each location;

R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group;

n is selected from 0, 1, 2, 3, 4 and 5; and

X is selected from —Br, —Cl, —F, —I.

In a preferred embodiment, at least two of H_(e), H_(f), and H_(g) arenot hydrogen. In another preferred embodiment, H_(g) is not R⁷(W)_(n).In another preferred embodiment, H_(g) is neither hydrogen norR⁷(W)_(n). In another preferred embodiment, none of H_(a), H_(b), H_(c),H_(d), H_(e), H_(f) or H_(g) is a heterocyclic ring.

In other aspects, in the compound of formula (3): H_(a) is hydrogen;H_(a) is not hydrogen, and the carbon to which H_(a) is bound has an Sconfiguration; H_(a) is not hydrogen and the carbon to which H_(a) isbound has an R configuration; H_(a) is —W; H_(a) is —R⁷(W)_(n); H_(a) is—CH₂-phenyl, and phenyl has 0, 1 or 2 W substitutions; H_(b) is W; H_(b)is —CN; H_(c) and H_(d) are both hydrogen; H_(e) is hydrogen; H_(e) ishydrogen and H_(f) is W; H_(e) is hydrogen and H_(f) is selected from—OH and —OR⁸; H_(e) is hydrogen and H_(f) is selected from methoxy,ethoxy, propoxy, cyclopentyloxy, cyclohexyloxy, and benzyloxy; H_(e) ishydrogen and H_(f) is selected from —NH₂, —NHR⁸, and —NR⁸R⁸; H_(g) ishydrogen; H_(g) is —W; H_(g) is selected from —OH and —OR⁸; H_(g) isselected from methoxy, ethoxy, propoxy, cyclopentyloxy, cyclohexyloxy,and benzyloxy; wherein H_(g) is selected from —NH₂, —NHR⁸, and —NR⁸R⁸;H_(g) is —R⁷(W)_(n); and/or H_(g) is C₁-C₃₀alkyl and n=0.

The compound of formula (3) may have the stereochemistry of formula (3a)

The compound of formula (3) may have the stereochemistry of formula (3b)

A reference herein to compounds of formula (3) includes reference tocompounds of formulae (3a) and (3b).

In another aspect, the present invention provides compounds of formula(4)

wherein, Q represents a multivalent atom other than carbon; and

each of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15,16, 17, 18, and 19 in formula (4), as well as Q to the extent that itmay be substituted, is independently substituted at each occurrence withH, —W or —R⁷(W)_(n), wherein

W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸,—OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸, —PR⁸R⁸, —POR⁸,—PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸, —SONR⁸R⁸, —SO₂NH₂,—SO₂NHR⁸ and —SO₂NR⁸R⁸;

R⁷ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group whereinn of the hydrogen or halogen atoms of R⁷are substituted by an equalnumber of W groups independently selected at each location;

R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group;

n is selected from 0, 1, 2, 3, 4 and 5; and

X is selected from —Br, —Cl, —F, —I.

In one aspect, the compound of formula (4) has the S configuration atcarbon 3. In another aspect, the compound of formula (4) has the Rconfiguration at carbon 3. In another aspect, the compound of formula(4) has the S configuration at carbon 5. In another aspect, the compoundof formula (4) has the R configuration at carbon 5. In another aspect,none of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15,16, 17, 18, or 19 in formula (4) is substituted with a heterocyclicmoiety.

In other aspects, in the compound of formula (4), as well as incompositions comprising the compound of formula (4) and apharmaceutically acceptable carrier, diluent or excipient: Q is O; Q isS; Q is NH; and/or Q is N(R⁷(W)_(n)). In other aspects, the carbon(s) atposition 4, or 6, and preferably both of positions 4 and 6, aresubstituted exclusively with hydrogen; the carbon at position 19 issubstituted with —W; the carbon at position 19 is substituted with —NH₂,—NHR⁸, or —NR⁸R⁸; the carbon at position 19 is substituted with —CN, —X,—OH, —NO₂, —SH, or —OR⁸; one carbon at positions 17 and 18 issubstituted with hydrogen; at least one carbon at positions 17 and 18 issubstituted with —W; at least one carbon at positions 17 and 18 issubstituted with —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸, or—OR⁸; at least one carbon at positions 17 and 18 is substituted with—NH₂, —NHR⁸, or —NR⁸R⁸; and/or at least one carbon at positions 17 and18 is substituted with —CN, —X, —OH, —NO₂, —SH, or —OR⁸. In anotheraspect, only one of the carbons at positions 17, 18 and 19 issubstituted with hydrogen. In another aspect, exactly two of the carbonsat positions 17, 18 and 19 are substituted with hydrogen. In anotheraspect, none of the carbons at positions 17, 18 and 19 are substitutedwith hydrogen. In another aspect, no more than one of the carbons atpositions 17, 18 and 19 are substituted with hydrogen.

In other aspects, in the compound of formula (4), as well as incompositions comprising the compound of formula (4) and apharmaceutically acceptable carrier, diluent or excipient: the carbon atposition 7 is substituted exclusively with hydrogen; the carbon atposition 3 is substituted with hydrogen; the carbon at position 3 issubstituted with —W; the carbon at position 3 is substituted withhalogen; the carbon at position 3 is substituted with —R⁷(W)_(n); thecarbon at position 3 is substituted with C₁-C₆hydrocarbyl; the carbonsat positions 9 and 10 are substituted with hydrogen; the carbons atpositions 11, 12, and 13 are independently substituted with hydrogen and—W; only one of the carbons at positions 11 and 12 is substituted withhydrogen; the carbon at position 11 and/or 12 is substituted with —NH₂,—CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂, —SH, —COX, —NHR⁸,—NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸, or —OR⁸; the carbon atposition 11 and/or 12 is substituted with —NH₂, —NHR⁸, or —NR⁸R⁸; thecarbon at position 11 and/or 12 is substituted with —CN, —X, —OH, —NO₂,—SH, or —OR⁸; the carbon at position 13 is substituted with —NH₂,—CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂, —SH, —COX, —NHR⁸,—NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸, or —OR⁸; the carbon atposition 13 is substituted with —NH₂, —NHR⁸, or —NR⁸R⁸; the carbon atposition 13 is substituted with —CN, —X, —OH, —NO₂, —SH, or —OR⁸; atleast one carbon from positions 11, 12, and 13 is substituted with—R⁷(W)_(n); and/or at least one carbon from positions 11, 12, and 13 issubstituted with C₁-C₆hydrocarbyl or C₁-C₆halocarbyl orC₁-C₆hydrohalocarbyl.

In another aspect, only one of the carbons at positions 11, 12, and 13is substituted with hydrogen. In another aspect, exactly two of thecarbons at positions 11, 12, and 13 are substituted with hydrogen. Inanother aspect, none of the carbons at positions 11, 12, and 13 aresubstituted with hydrogen. In another aspect, no more than one of thecarbons at positions 11, 12, and 13 are substituted with hydrogen.

In compounds of formula (4), and compositions comprising one or morecompounds of formula (4) and a pharmaceutically acceptable carrier,diluent or excipient, the carbon at position 6 is preferably notsubstituted with either ═O or ═S; the carbon at position 4 is preferablynot substituted with ═O; the phenyl ring bonded to the carbon atposition 5 is preferably substituted with no more than 4 hydrogen atoms;the phenyl ring bonded to the carbon at position 5 is preferablysubstituted with no more than 4 R⁷(W)_(n) groups, and/or the compoundsof formula (4) preferably exclude massonianalactone.

Thus, in a preferred compound of formula (4), Q is O or NH, the carbonat position 6 is substituted with not substituted with either ═O or ═S;the carbon at position 4 is not substituted with ═O; the phenyl ringbonded directly to carbon 5 is directly substituted in at least oneposition with an atom other than carbon or hydrogen; andmassonianalactone is excluded. Massonianalactone, which has the CASRegistry No. of 150270-05-6, is also known as 2H-pyran-2-one,tetrahydro-3-hydroxy-5-(4-hydroxy-3-methoxyphenyl)-3-[(4-hydroxy-3-methoxyphenyl)methyl]-,(3R-trans).

In a preferred embodiment, in compounds of formula (4), and compositionscomprising a compound of formula (4), Q is NH, and not both of positions17 and 18 are substituted with hydrogen.

For example, the present invention provides a Δ-lactam compound, and inparticular a γ-Phenyl-substituted Δ-lactam compound of the formula (4)wherein Q is N or substituted N, as follows:

wherein each of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 15, 16, 17, 18, and 19, as well as the nitrogen at position 1, isindependently substituted at each occurrence with H, —W or —R⁷(W)_(n),

W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸,—OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸, —PR⁸R⁸, —POR⁸,—PO₂R⁸, —PO₃R⁸, —SR⁸, —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸, —SONR⁸R⁸, —SO₂NH₂,—SO₂NHR⁸ and —SO₂NR⁸R⁸;

R⁷ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group whereinn of the hydrogen or halogen atoms of R⁷ are substituted by an equalnumber of W groups independently selected at each location;

R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group;

n is selected from 0, 1, 2, 3, 4 and 5; and

X is selected from —Br, —Cl, —F, and —I.

In various embodiments of the aspect of the present invention thatprovides a γ-Phenyl-substituted Δ-lactam compound of the formula (4)wherein Q is N or substituted N, the following criteria may be usedalone or in any combination in further describing the compound of theinvention, where these criteria are exemplary only and other criteriamay be found elsewhere herein: positions 4 and 6 are substitutedexclusively with hydrogen; position 19 is substituted with —W; position19 is substituted wit; —CN, —X, —OH, —NO₂, —SH, or —OR⁸; one carbon atpositions 17 and 18 is substituted with hydrogen; at least one carbon atpositions 17 and 18 is substituted with —W; at least one carbon atpositions 17 and 18 is substituted with —NH₂, —CONH₂, —COOH, —CN, —CHO,—OCHO, —X, —OH, —NO₂, —SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸,—COOR⁸, —COR⁸, —OCOR⁸, or —OR⁸; at least one carbon at positions 17 and18 is substituted with —CN, —X, —OH, —NO₂, —SH, or —OR⁸; only one of thecarbons at positions 17, 18 and 19 is substituted with hydrogen; thecarbon at position 7 is substituted exclusively with hydrogen; thecarbon at position 3 is substituted with hydrogen, R⁸ or X; the carbonsat positions 9 and 10 are substituted with hydrogen; the carbons atpositions 11, 12, and 13 are independently substituted with hydrogen and—W; only one of the carbons at positions 11 and 12 is substituted withhydrogen; a carbon selected from positions 11 and 12 is substituted with—CN, —X, —OH, —NO₂, —SH, or —OR⁸; at least one carbon from positions 11,12, and 13 is substituted with —R⁷(W)_(n); at least one carbon frompositions 11, 12, and 13 is substituted with C₁-C₆hydrocarbyl,C₁-C₆halocarbyl or C₁-C₆hydrohalocarbyl; exactly two of the carbons atpositions 11, 12 and 13 are substituted with hydrogen; W is selectedfrom, —OCHO, —OH, —OCOR⁸, and —OR⁸; W is selected from —NH₂, —CN, —X,—OH, —NO₂, —SH, —NHR⁸, —NR⁸R⁸, —OR⁸, and —SR⁸; W is —OR⁸; R⁷ is a C₁-C₁₀hydrocarbyl group wherein n of the hydrogen or halogen atoms of R⁷ aresubstituted by an equal number of W groups independently selected ateach location; R⁷ is selected from the group consisting of alkyl,alkenyl, alkynyl, aryl, aralkyl, alkylaryl, alkenyl-substituted aryl,aryl-substituted alkenyl, alkynyl-substituted aryl, aryl-substitutedalkynyl, biaryl, cycloalkyl, cycloalkenyl, bicycloalkyl, bicycloalkenyl,alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, aryl-substitutedcycloalkyl, cycloalkyl-substituted aryl, aryl-substituted cycloalkenyl,cycloalkenyl-substituted aryl, aryl-fused cycloalkyl and polycycloalkyl;R⁸ is a C₁-C₁₀ hydrocarbyl group; R⁸ is a C₁-C₁₀ cyclohydrocarbyl group;R⁸ is selected from the group consisting of alkyl, alkenyl, alkynyl,aryl, aralkyl, alkylaryl, alkenyl-substituted aryl, aryl-substitutedalkenyl, alkynyl-substituted aryl, aryl-substituted alkynyl, biaryl,cycloalkyl, cycloalkenyl, bicycloalkyl, bicycloalkenyl, alkylcycloalkyl,alkenylcycloalkyl, alkynylcycloalkyl, aryl-substituted cycloalkyl,cycloalkyl-substituted aryl, aryl-substituted cycloalkenyl,cycloalkenyl-substituted aryl, aryl-fused cycloalkyl and polycycloalkyl;n is 0; position 1 is substituted with hydrogen; carbon 3 has the Sconfiguration; carbon 3 has the R configuration; carbon 5 has the Sconfiguration; carbon 5 has the the R configuration; wherein positions3, 4, 6, 7, 9 and 10 are substituted with hydrogen, one of positions 11and 12 is substituted with hydrogen, and one of positions 17 and 18 issubstituted with hydrogen; at least one carbon from positions 11, 12,and 13 is substituted with C₁-C₆hydrocarbyl, C₁-C₆halocarbyl orC₁-C₆hydrohalocarbyl, and exactly two of the carbons at positions 11, 12and 13 are substituted with hydrogen; position 19 is substituted with—CN, —X, —OH, —NO₂, —SH, or —OR⁸ and one carbon at positions 17 and 18is substituted with hydrogen and at least one carbon at positions 17 and18 is substituted with —CN, —X, —OH, —NO₂, —SH, or —OR⁸; positions 3, 4,6, 7, 9 and 10 are substituted with hydrogen, and exactly two of thecarbons at positions I1, 12 and 13 are substituted with hydrogen, and atleast one carbon from positions 11, 12, and 13 is substituted withC₁-C₆hydrocarbyl, C₁-C₆halocarbyl or C₁-C₆hydrohalocarbyl, and one ofpositions 17 and 18 is substituted with hydrogen, and position 19 issubstituted with —CN, —X, —OH, —NO₂, —SH, or —OR⁸, and at least onecarbon at positions 17 and 18 is substituted with —CN, —X, —OH, —NO₂,—SH, or —OR⁸, and one carbon at positions 17 and 18 is substituted withhydrogen; positions 1, 3, 4, 6, 7, 9, 10, 11 and 18 are substituted withhydrogen, and positions 17 and 19 are substituted with OR⁸ where R⁸ is aC₁-C₁₀ hydrocarbyl group, and position 12 is substituted withC₁-C₆hydrocarbyl, C₁-C₆halocarbyl or C₁-C₆hydrohalocarbyl; positions 1,3, 4, 5, 6, 7, 9, 10, 11, 13, 15, 16, and 18 are substituted withhydrogen, and position 12 is substituted with C₁ hydrocarbyl, andposition 17 is substituted with —O-cyclopentyl, and position 19 issubstituted with —O-methyl, where optionally positions 3 and 5 both havethe S stereochemistry. Any of these compounds may be in the form of asalt or solvate according to the present invention.

The compounds disclosed herein of formulae 1, 2, 3 or 4 (i.e., compoundsof formulae (1-4), or compounds of the present invention), orcompositions comprising one of more of these compounds and apharmaceutically acceptable carrier, diluent or excipient, may be usedin a method for treating or preventing an inflammatory condition ordisease in a patient, where the method comprises administering to thepatient in need thereof an amount of a compound or composition accordingto the present invention, where the amount is effective to treat orprevent the inflammatory condition or disease of the patient.

The inflammatory condition or disease may be an autoimmune condition ordisease; the inflammatory condition or disease may involve acute orchronic inflammation of bone and/or cartilage compartments of joints;the inflammatory condition or disease may be an arthritis selected fromrheumatoid arthritis, gouty arthritis or juvenile rheumatoid arthritis;the inflammatory condition or disease may be asthma; the condition ordisease may be associated with the disregulation of T-cells; thecondition or disease may be associated with elevated levels ofinflammatory cytokines (e.g., wherein the inflammatory cytokine is IL-2,or wherein the inflammatory cytokine is IFN-γ, or wherein theinflammatory cytokine is TNF-α); the inflammatory condition or diseasemay be multiple sclerosis; the inflammatory condition or disease may bepulmonary sarcadosis; the inflammatory condition or disease may beocular inflammation or allergy; the inflammatory condition or diseasemay be an inflammatory bowel disease (e.g., Crohn's disease orulcerative colitis); and the inflammatory condition or disease may be aninflammatory cutaneous disease (e.g., psoriasis or dermatitis).

Furthermore, the present invention provides a method for modulatingintracellular cyclic adenosine 5′-monophosphate levels within a patient,comprising administering to a patient in need thereof an amount of acompound or composition according to the present invention, wherein theamount is effective to modulate the intracellular cyclic adenosine5′-monophosphate levels of the patient. The patient may have aninflammatory condition or disease.

Furthermore, the present invention provides a method for treating orpreventing a disease or condition in a patient, where the disease orcondition is associated with pathological conditions that are modulatedby inhibiting enzymes associated with secondary cellular messengers, themethod comprising administering to a patient in need thereof an amountof a compound or a composition of the present invention, wherein theamount is effective to treat or prevent a disease or conditionassociated with pathological conditions that are modulated by inhibitingenzymes associated with secondary cellular messengers. The enzyme may bea cyclic AMP phosphodiesterase; or the enzyme may be a phosphodiesterase4; or the enzyme may be a phosphodiesterase 3; or the enzymes may beboth of phosphodiesterase 4 and phosphodiesterase 3; or the enzyme maybe a cyclic GMP phosphodiesterase.

Furthermore, the present invention provides a method of treating orpreventing transplant rejection in a patient, comprising administeringto a patient in need thereof an amount of a compound or composition ofthe present invention, where the amount is effective to treat or preventtransplant rejection in the patient. The rejection may be due to graftversus host disease.

Furthermore, the present invention provides a method of treating orpreventing uncontrolled cellular proliferation in a patient, comprisingadministering to a patient in need thereof an amount of a compound orcomposition according to the present invention, where the amount iseffective to treat or prevent uncontrolled cellular proliferation in thepatient. The uncontrolled cellular proliferation may be caused by acancer selected from leukemia and solid tumors.

Furthermore, the present invention provides a method of treating orpreventing conditions associated with the central nervous system (CNS)in a patient, comprising administering to a patient in need thereof anamount of a compound or composition according to the present invention,where the amount is effective to treat or prevent conditions associatedwith the central nervous system (CNS) in the patient. The conditionassociated with the central nervous system (CNS) may be depression.

In a method of the present invention, a compound of formulae (1-4), or acomposition comprising one or more compounds of formulae (1-4) and apharmaceutically acceptable carrier, diluent or excipient, may, althoughneed not, achieve one or more of the following desired results in thesubject to whom has been administered a compound of formulae (1-4) asdefined above, or a composition containing one of these compounds and apharmaceutically acceptable carrier, diluent or excipient:

1. Inhibition of reactive oxygen species generation from primaryneutrophils;

2. Inhibition of neutrophil chemotaxis;

3. Inhibition of TNF-α production;

4. Inhibition of edema;

5. Oxygen radical scavenging;

6. Inhibition of cyclic-AMP phosphodiesterases 1, 3 and/or 4, andrelated PDEs such as PDE7;

7. Potentiate induction of CRE-mediated transcription activity in humanmonocytic cells;

8. Inhibition of PDE, preferably PDE4, PDE3, or PDE3 and PDE4;

9. Inhibition of cytokine production by activated T-cell subsets;

10. Inhibition of neutrophil myeloperoxidase release;

11. Low ratio of IC₅₀PDE4(cat):IC₅₀PDE4(HARBS);

12. Inhibition of graft rejection;

13. Inhibition of clinical and histopathological parameters of diseasein inflammatory bowel disease; and

14. Inhibition of clinical and histopathological parameters of arthritisin a murine collage-induced arthritis model.

These and other aspects and embodiments of the present invention will beapparent upon reference to the following detailed description. To thisend, various references are set forth herein which describe in moredetail certain procedures, compounds and/or compositions, and are herebyincorporated by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds, compositions and methodsuseful in the treatment and/or prevent of various disease conditions.For example, in one aspect, the present invention provides a method oftreating and/or preventing an inflammatory disease. The method includesadministering to a subject in need thereof a therapeutically-effectiveamount of a compound or a pharmaceutically acceptable salt thereof, or atherapeutically effective amount of a composition containing a compoundof formulae or a pharmaceutically acceptable salt thereof, of any of thecompounds of formulae (1-4) as defined herein.

In one aspect, the present invention provides a composition comprising acompound according to formula (1) and salts, solvates, isolatedstereoisomers, and mixtures thereof, and a pharmaceutically acceptablecarrier, diluent, or excipient,

wherein each of hydrogens H_(a), H_(b), H_(c), H_(d), H_(e), H_(f) andH_(g) may independently be replaced with a group selected from —W and—R⁷(W)_(n), and M₁ represents —W or —R⁷(W)_(n), wherein

W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸,—OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸, —PR⁸R⁸, —POR⁸,—PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸, —SONR⁸R⁸, —SO₂NH₂,—SO₂NHR⁸ and —SO₂NR⁸R⁸;

R⁷ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group whereinn of the hydrogen or halogen atoms of R⁷ are substituted by an equalnumber of W groups independently selected at each location;

R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group;

n is selected from 0, 1, 2, 3, 4 and 5; and

X is selected from —Br, —Cl, —F, —I.

In other aspects of the composition comprising a compound of formula(1): H_(a) and H_(b) are hydrogen; H_(a) is hydrogen and H_(b) is —W;H_(a) is hydrogen, H_(b) is —W and the carbon to which H_(b) is boundhas an S configuration; H_(a) is hydrogen, H_(b) is —W, and the carbonto which H_(b) is bound has an R configuration; H_(a) is hydrogen andH_(b) is —R⁷(W)_(n); H_(a) is hydrogen, H_(b) is —R⁷(W)_(n), and thecarbon to which H_(b) is bound has an S configuration; H_(a) ishydrogen, H_(b) is —R⁷(W)_(n), and the carbon to which H_(b) is boundhas an R configuration; H_(a) is hydrogen, H_(b) is —R⁷(W)_(n), H_(b) is—CH₂-phenyl, and phenyl has 0, 1 or 2 W substitutions; H_(c) is W; H_(d)and H_(e) are both hydrogen; H_(f) is W; H_(f) is selected from —OH and—OR⁸; H_(f) is selected from methoxy, ethoxy, propoxy, cyclopentyloxy,cyclohexyloxy, and benzyloxy; H_(f) is selected from —NH₂, —NHR⁸, and—NR⁸R⁸; H_(g) is —R⁷(W)_(n); M₁ is —W; M₁ is selected from methoxy,ethoxy, propoxy, cyclopentyloxy, cyclohexyloxy, and benzyloxy; M₁ isselected from —NH₂, —NHR⁸, and —NR⁸R⁸; M₁ is selected from —OH and —OR⁸;and/or M₁ is —R⁷(W)_(n). In one embodiment, none of H_(a), H_(b), H_(c),H_(d), H_(e), H_(f) or H_(g) is a heterocyclic ring.

The compound of formula (1) may have the stereochemistry of formula (1a)

The compound of formula (1) may have the stereochemistry of formula (1b)

A reference herein to compounds of formula (1) includes compounds offormulae (1a) and (1b).

In another aspect, the present invention provides a compositioncomprising a compound according to formula (2) and salts, solvates,isolated stereoisomers, and mixtures thereof, and a pharmaceuticallyacceptable carrier, diluent, or excipient,

wherein each of hydrogens H_(a), H_(b), H_(c), H_(d), H_(e), H_(f) andH_(g) may independently be replaced with a group selected from —W and—R⁷(W)_(n), and M₂ represents —W, wherein

W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸,—OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸, —PR⁸R⁸, —POR⁸,—PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸, —SONR⁸R⁸, —SO₂NH₂,—SO₂NHR⁸ and —SO₂NR⁸R⁸;

R⁷ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group whereinn of the hydrogen or halogen atoms of R⁷ are substituted by an equalnumber of W groups independently selected at each location;

R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group;

n is selected from 0, 1, 2, 3, 4 and 5; and

X is selected from —Br, —Cl, —F, —I.

In other aspects of compositions comprising a compound of formula (2):H_(a) and H_(b) are hydrogen; H_(a) is hydrogen and H_(b) is —W; H_(a)is hydrogen, H_(b) is —W, and the carbon to which H_(b) is bound has anS configuration; H_(a) is hydrogen, H_(b) is —W, and the carbon to whichH_(b) is bound has an R configuration; H_(a) is hydrogen and H_(b) is—R⁷(W)_(n); H_(a) is hydrogen, H_(b) is —R⁷(W)_(n), and the carbon towhich H_(b) is bound has an S configuration; H_(a) is hydrogen, H_(b) is—R⁷(W)_(n), and the carbon to which H_(b) is bound has an rconfiguration; H_(a) is hydrogen, and H_(b) is —CH₂-phenyl, where phenylhas 0, 1 or 2 W substitutions; H, is W; H_(d) and H_(e) are bothhydrogen; H_(f) is hydrogen and H_(g) is W; H_(g) is selected from —OHand —OR⁸; H_(g) is selected from methoxy, ethoxy, propoxy,cyclopentyloxy, cyclohexyloxy, and benzyloxy; H_(g) is selected from—NH₂, —NHR⁸, and —NR⁸R⁸; H_(f) is hydrogen and H_(g) is —R⁷(W)_(n); M₂is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸, and—OR⁸; and/or M₂ is selected from —NH₂, —NHR⁸, —NR⁸R⁸, —OH, and —OR⁸. Ina preferred embodiment, none of H_(a), H_(b), H_(c), H_(d), H_(c), H_(f)or H_(g) may be replaced with a heterocyclic ring system.

The compound of formula (2) may have the stereochemistry of formula (2a)

The compound of formula (2) may have the stereochemistry of formula (2b)

A reference herein to compounds of formula (2) includes reference tocompounds of formulae (2a) and (2b).

In another aspect, the present invention provides a compound accordingto formula (3) and salts, solvates, isolated stereoisomers, and mixturesthereof,

wherein each of hydrogens H_(a), H_(c), H_(d), H_(e), H_(f) and H_(g)may independently be replaced with a group selected from —W and—R⁷(W)_(n), and H_(b) may be replaced with —W, wherein

W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸,—OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸, —PR⁸R⁸, —POR⁸,—PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸, —SONR⁸R⁸, —SO₂NH₂,—SO₂NHR⁸ and —SO₂NR⁸R⁸;

R⁷ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group whereinn of the hydrogen or halogen atoms of R⁷ are substituted by an equalnumber of W groups independently selected at each location;

R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group;

n is selected from 0, 1, 2, 3, 4 and 5; and

X is selected from —Br, —Cl, —F, —I.

In a preferred embodiment, at least two of H_(e), H_(f), and H_(g) arenot hydrogen. In another preferred embodiment, H_(g) is not R⁷(W)_(n).In another preferred embodiment, H_(g) is neither hydrogen norR⁷(W)_(n). In another preferred embodiment, none of H_(a), H_(b), H_(c),H_(d), H_(e), H_(f) or H_(g) is a heterocyclic ring.

In other aspects, in the compound of formula (3): H_(a) is hydrogen;H_(a) is not hydrogen, and the carbon to which H_(a) is bound has an Sconfiguration; H_(a) is not hydrogen and the carbon to which H_(a) isbound has an R configuration; H_(a) is —W; H_(a) is —R⁷(W)_(n); H_(a) is—CH₂-phenyl, and phenyl has 0, 1 or 2 W substitutions; H_(b) is W; H_(b)is —CN; H_(c) and H_(d) are both hydrogen; H_(e) is hydrogen; H_(e) ishydrogen and H_(f) is W; H_(e) is hydrogen and H_(f) is selected from—OH and —OR⁸; H_(e) is hydrogen and H_(f) is selected from methoxy,ethoxy, propoxy, cyclopentyloxy, cyclohexyloxy, and benzyloxy; H_(e) ishydrogen and H_(f) is selected from —NH₂, —NHR⁸, and —NR⁸R⁸; H_(g) ishydrogen; H_(g) is —W; H_(g) is selected from —OH and —OR⁸; H_(g) isselected from methoxy, ethoxy, propoxy, cyclopentyloxy, cyclohexyloxy,and benzyloxy; wherein H_(g) is selected from —NH₂, —NHR⁸, and —NR⁸R⁸;H_(g) is —R⁷(W)_(n); and/or H_(g) is C₁-C₃₀alkyl and n=0.

The compound of formula (3) may have the stereochemistry of formula (3a)

The compound of formula (3) may have the stereochemistry of formula (3b)

A reference herein to compounds of formula (3) includes reference tocompounds of formulae (3a) and (3b).

In another aspect, the present invention provides compounds of formula(4)

wherein, Q represents a multivalent atom other than carbon; and

each of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15,16, 17, 18, and 19 in formula (4), as well as Q to the extent that itmay be substituted, is independently substituted at each occurrence withH, —W or —R ⁷(W)_(n), wherein

W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸, OR⁸,—BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸, —PR⁸R⁸, —POR⁸,—PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸, —SONR⁸R⁸, —SO₂NH₂,—SO₂NHR⁸ and —SO₂NR⁸R⁸;

R⁷ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group whereinn of the hydrogen or halogen atoms of R⁷ are substituted by an equalnumber of W groups independently selected at each location;

R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group;

n is selected from 0, 1, 2, 3, 4 and 5; and

X is selected from —Br, —Cl, —F, —I.

In one aspect, the compound of formula (4) has the S configuration atcarbon 3. In another aspect, the compound of formula (4) has the Rconfiguration at carbon 3. In another aspect, the compound of formula(4) has the S configuration at carbon 5. In another aspect, the compoundof formula (4) has the R configuration at carbon 5. In another aspect,none of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 15,16, 17, 18, or 19 in formula (4) is substituted with a heterocyclicmoiety.

In other aspects, in the compound of formula (4), as well as incompositions comprising the compound of formula (4) and apharmaceutically acceptable carrier, diluent or excipient: Q is O; Q isS; Q is NH; and/or Q is N(R⁷(W)_(n)). In other aspects, the carbon(s) atposition 4, or 6, and preferably both of positions 4 and 6, aresubstituted exclusively with hydrogen; the carbon at position 19 issubstituted with —W; the carbon at position 19 is substituted with —NH₂,—NHR⁸, or —NR⁸R⁸; the carbon at position 19 is substituted with —CN, —X,—OH, —NO₂, —SH, or —OR⁸; one carbon at positions 17 and 18 issubstituted with hydrogen; at least one carbon at positions 17 and 18 issubstituted with —W; at least one carbon at positions 17 and 18 issubstituted with —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸, or—OR⁸; at least one carbon at positions 17 and 18 is substituted with—NH₂, —NHR⁸, or —NR⁸R⁸; and/or at least one carbon at positions 17 and18 is substituted with —CN, —X, —OH, —NO₂, —SH, or —OR⁸. In anotheraspect, only one of the carbons at positions 17, 18 and 19 issubstituted with hydrogen. In another aspect, exactly two of the carbonsat positions 17, 18 and 19 are substituted with hydrogen. In anotheraspect, none of the carbons at positions 17, 18 and 19 are substitutedwith hydrogen. In another aspect, no more than one of the carbons atpositions 17, 18 and 19 are substituted with hydrogen.

In other aspects, in the compound of formula (4), as well as incompositions comprising the compound of formula (4) and apharmaceutically acceptable carrier, diluent or excipient: the carbon atposition 7 is substituted exclusively with hydrogen; the carbon atposition 3 is substituted with hydrogen; the carbon at position 3 issubstituted with —W; the carbon at position 3 is substituted withhalogen; the carbon at position 3 is substituted with —R⁷(W)_(n); thecarbon at position 3 is substituted with C₁-C₆hydrocarbyl; the carbonsat positions 9 and 10 are substituted with hydrogen; the carbons atpositions 11, 12, and 13 are independently substituted with hydrogen and—W; only one of the carbons at positions 11 and 12 is substituted withhydrogen; the carbon at position 11 and/or 12 is substituted with —NH₂,—CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂, —SH, —COX, —NHR⁸,—NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸, or —OR⁸; the carbon atposition 11 and/or 12 is substituted with —NH₂, —NHR⁸, or —NR⁸R⁸; thecarbon at position 11 and/or 12 is substituted with —CN, —X, —OH, —NO₂,—SH, or —OR⁸; the carbon at position 13 is substituted with —NH₂,—CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂, —SH, —COX, —NHR⁸,—NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸, or —OR⁸; the carbon atposition 13 is substituted with —NH₂, —NHR⁸, or —NR⁸R⁸; the carbon atposition 13 is substituted with —CN, —X, —OH, —NO₂, —SH, or —OR⁸; atleast one carbon from positions 11, 12, and 13 is substituted with—R⁷(W)_(n);

In another aspect, only one of the carbons at positions 11, 12, and 13is substituted with hydrogen. In another aspect, exactly two of thecarbons at positions 11, 12, and 13 are substituted with hydrogen. Inanother aspect, none of the carbons at positions 11, 12, and 13 aresubstituted with hydrogen. In another aspect, no more than one of thecarbons at positions 11, 12, and 13 are substituted with hydrogen.

In compounds of formula (4), and compositions comprising one or morecompounds of formula (4) and a pharmaceutically acceptable carrier,diluent or excipient, the carbon at position 6 is preferably notsubstituted with either ═O or ═S; the carbon at position 4 is preferablynot substituted with ═O; the phenyl ring bonded to the carbon atposition 5 is preferably substituted with no more than 4 hydrogen atoms;the phenyl ring bonded to the carbon at position 5 is preferablysubstituted with no more than 4 R⁷(W)_(n) groups, and/or the compoundsof formula (4) preferably exclude massonianalactone.

Thus, in a preferred compound of formula (4), Q is O or NH, the carbonat position 6 is not substituted with either ═O or ═S; the carbon atposition 4 is not substituted with ═O; the phenyl ring bonded directlyto carbon 5 is directly substituted in at least one position with anatom other than carbon or hydrogen; and massonianalactone is excluded.Massonianalactone, which has the CAS Registry No. of 150270-05-6, isalso known as 2H-pyran-2-one,tetrahydro-3-hydroxy-5-(4-hydroxy-3-methoxyphenyl)-3-[(4-hydroxy-3-methoxyphenyl)methyl]-,(3R-trans).

For example, the present invention provides a Δ-lactam compound, and inparticular a γ-Phenyl-substituted Δ-lactam compound of the formula (4)wherein Q is N or substituted N, as follows:

wherein each of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 15, 16, 17, 18, and 19, as well as the nitrogen at position 1, isindependently substituted at each occurrence with H, —W or —R⁷(W)_(n),

W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH, —NO₂,—SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸, —OCOR⁸,—OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸, —PR⁸R⁸, —POR⁸,—PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸, —SONR⁸R⁸, —SO₂NH₂,—SO₂NHR⁸ and —SO₂NR⁸R⁸;

R⁷ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group whereinn of the hydrogen or halogen atoms of R⁷ are substituted by an equalnumber of W groups independently selected at each location;

R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group;

n is selected from 0, 1, 2, 3, 4 and 5; and

X is selected from —Br, —Cl, —F, and —I.

In various embodiments of the aspect of the present invention thatprovides a γ-Phenyl-substituted Δ-lactam compound of the formula (4)wherein Q is N or substituted N, the following criteria may be usedalone or in any combination in further describing the compound(s) of theinvention, where these criteria are exemplary only and other criteriamay be found elsewhere herein: positions 4 and 6 are substitutedexclusively with hydrogen; position 19 is substituted with —W; position19 is substituted with —CN, —X, —OH, —NO₂, —SH, or —OR⁸; one carbon atpositions 17 and 18 is substituted with hydrogen; at least one carbon atpositions 17 and 18 is substituted with —W; at least one carbon atpositions 17 and 18 is substituted with —NH₂, —CONH₂, —COOH, —CN, —CHO,—OCHO, —X, —OH, —NO₂, —SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸,—COOR⁸, —COR⁸, —OCOR⁸, or —OR⁸; at least one carbon at positions 17 and18 is substituted with —CN, —X, —OH, —NO₂, —SH, or —OR⁸; only one of thecarbons at positions 17, 18 and 19 is substituted with hydrogen; thecarbon at position 7 is substituted exclusively with hydrogen; thecarbon at position 3 is substituted with hydrogen, R⁸ or X; the carbonsat positions 9 and 10 are substituted with hydrogen; the carbons atpositions 11, 12, and 13 are independently substituted with hydrogen and—W; only one of the carbons at positions 11 and 12 is substituted withhydrogen; a carbon selected from positions 11 and 12 is substituted with—CN, —X, —OH, —NO₂, —SH, or —OR⁸; at least one carbon from positions 11,12, and 13 is substituted with —R⁷(W)_(n); at least one carbon frompositions 11, 12, and 13 is substituted with C₁-C₆hydrocarbyl,C₁-C₆halocarbyl or C₁-C₆hydrohalocarbyl; exactly two of the carbons atpositions 11, 12 and 13 are substituted with hydrogen; W is selectedfrom, —OCHO, —OH, —OCOR⁸, and —OR⁸; W is selected from —NH₂, —CN, —X,—OH, —NO₂, —SH, —NHR⁸, —NR⁸R⁸, —OR⁸, and —SR⁸; W is —OR⁸; R⁷ is a C₁-C₁₀hydrocarbyl group wherein n of the hydrogen or halogen atoms of R⁷ aresubstituted by an equal number of W groups independently selected ateach location; R⁷ is selected from the group consisting of alkyl,alkenyl, alkynyl, aryl, aralkyl, alkylaryl, alkenyl-substituted aryl,aryl-substituted alkenyl, alkynyl-substituted aryl, aryl-substitutedalkynyl, biaryl, cycloalkyl, cycloalkenyl, bicycloalkyl, bicycloalkenyl,alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, aryl-substitutedcycloalkyl, cycloalkyl-substituted aryl, aryl-substituted cycloalkenyl,cycloalkenyl-substituted aryl, aryl-fused cycloalkyl and polycycloalkyl;R⁸ is a C₁-C₁₀ hydrocarbyl group; R⁸ is a C₁-C₁₀ cyclohydrocarbyl group;R⁸ is selected from the group consisting of alkyl, alkenyl, alkynyl,aryl, aralkyl, alkylaryl, alkenyl-substituted aryl, aryl-substitutedalkenyl, alkynyl-substituted aryl, aryl-substituted alkynyl, biaryl,cycloalkyl, cycloalkenyl, bicycloalkyl, bicycloalkenyl, alkylcycloalkyl,alkenylcycloalkyl, alkynylcycloalkyl, aryl-substituted cycloalkyl,cycloalkyl-substituted aryl, aryl-substituted cycloalkenyl,cycloalkenyl-substituted aryl, aryl-fused cycloalkyl and polycycloalkyl;n is 0; position 1 is substituted with hydrogen; carbon 3 has the Sconfiguration; carbon 3 has the R configuration; carbon 5 has the Sconfiguration; carbon 5 has the the R configuration; wherein positions3, 4, 6, 7, 9 and 10 are substituted with hydrogen, one of positions 11and 12 is substituted with hydrogen, and one of positions 17 and 18 issubstituted with hydrogen; at least one carbon from positions 11, 12,and 13 is substituted with C₁-C₆hydrocarbyl, C₁-C₆halocarbyl orC₁-C₆hydrohalocarbyl, and exactly two of the carbons at positions 11, 12and 13 are substituted with hydrogen; position 19 is substituted with—CN, —X, —OH, —NO₂, —SH, or —OR⁸ and one carbon at positions 17 and 18is substituted with hydrogen and at least one carbon at positions 17 and18 is substituted with —CN, —X, —OH, —NO₂, —SH, or —OR⁸; positions 3, 4,6, 7, 9 and 10 are substituted with hydrogen, and exactly two of thecarbons at positions 11, 12 and 13 are substituted with hydrogen, and atleast one carbon from positions 11, 12, and 13 is substituted withC₁-C₆hydrocarbyl, C₁-C₆halocarbyl or C₁-C₆hydrohalocarbyl, and one ofpositions 17 and 18 is substituted with hydrogen, and position 19 issubstituted with —CN, —X, —OH, —NO₂, —SH, or —OR⁸, and at least onecarbon at positions 17 and 18 is substituted with —CN, —X, —OH, —NO₂,—SH, or —OR⁸, and one carbon at positions 17 and 18 is substituted withhydrogen; positions 1, 3, 4, 6, 7, 9, 10, 11 and 18 are substituted withhydrogen, and positions 17 and 19 are substituted with OR⁸ where R⁸ is aC_(1-C) ₁₀ hydrocarbyl group, and position 12 is substituted withC₁-C₆hydrocarbyl, C₁-C₆halocarbyl or C₁-C₆hydrohalocarbyl; positions 1,3, 4, 5, 6, 7, 9, 10, 11, 13, 15, 16, and 18 are substituted withhydrogen, and position 12 is substituted with C₁ hydrocarbyl, andposition 17 is substituted with —O-cyclopentyl; and position 19 issubstituted with —O-methyl, where optionally positions 3 and 5 both havethe S stereochemistry. Any of these compounds may be in the form of apharmaceutically acceptable salt or solvate according to the presentinvention.

In the γ-Phenyl-substituted Δ-lactam compounds of the formula (4)wherein Q is N or substituted N, the ring containing Q is monocyclic andsaturated, as shown in the formula (4).

In compounds of formulae (1-4):

W may be selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH,—NO₂, —SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸,—OCOR⁸, —OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, PHR⁸, —PR⁸R⁸,—POR⁸, —PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸, —SONR⁸R⁸,—SO₂NH₂, —SO₂NHR⁸ and —SO₂NR⁸R⁸;

R⁷ may be a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl groupwherein n of the hydrogen or halogen atoms of R⁷ are substituted by anequal number of W groups independently selected at each location;

R⁸ may be a C₁-C₃₀ hydrocarbyl, halocarbyl or hydrohalocarbyl group;

n may be selected from 0, 1, 2, 3, 4 and 5; and

X may be selected from —Br, —Cl, —F, —I.

In preferred embodiments: W is selected from —NH₂, —NHR⁸, and —NR⁸R⁸; Wis selected from —CONH₂, —COOH, —CN, —CHO, —COX, —CONHR⁸, —CONR⁸R⁸,—COOR⁸, —COR⁸; W is selected from, —OCHO, —OH, —OCOR⁸, and —OR⁸; W isselected from —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸,—PR⁸R⁸, —POR⁸, —PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸,—SONR⁸R⁸, —SO₂NH₂, —SO₂NHR⁸ and —SO₂NR⁸R⁸; W is selected from —NH₂, —CN,—X, —OH, —NO₂, —SH, —NHR⁸, —NR⁸R⁸, —OR⁸, and —SR⁸; and W is —OR⁸.

In preferred embodiments: R⁷ is a C₁-C₃₀ hydrocarbyl group wherein n ofthe hydrogen or halogen atoms of R⁷ are substituted by an equal numberof W groups independently selected at each location; R⁷ is a C₁-C₁₀hydrocarbyl group wherein n of the hydrogen or halogen atoms of R⁷ aresubstituted by an equal number of W groups independently selected ateach location; and/or R⁷ is selected from alkyl, alkenyl, alkynyl, aryl,aralkyl, alkylaryl, alkenyl-substituted aryl, aryl-substituted alkenyl,alkynyl-substituted aryl, aryl-substituted alkynyl, biaryl, cycloalkyl,cycloalkenyl, bicycloalkyl, bicycloalkenyl, alkylcycloalkyl,alkenylcycloalkyl, alkynylcycloalkyl, aryl-substituted cycloalkyl,cycloalkyl-substituted aryl, aryl-substituted cycloalkenyl,cycloalkenyl-substituted aryl, aryl-fused cycloalkyl and polycycloalkyl.

In preferred embodiments: R⁸ is a C₁-C₃₀ hydrocarbyl group; R⁸ is aC₁-C₁₀ hydrocarbyl group; and/or R⁸ is selected from alkyl, alkenyl,alkynyl, aryl, aralkyl, alkylaryl, alkenyl-substituted aryl,aryl-substituted alkenyl, alkynyl-substituted aryl, aryl-substitutedalkynyl, biaryl, cycloalkyl, cycloalkenyl, bicycloalkyl, bicycloalkenyl,alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, aryl-substitutedcycloalkyl, cycloalkyl-substituted aryl, aryl-substituted cycloalkenyl,cycloalkenyl-substituted aryl, aryl-fused cycloalkyl and polycycloalkyl.

In preferred embodiments: n is 0; n is 1; n is 2; n is 3; n is 4; n is5; n is greater than 0; n is 1 or 2; and n is 1 or 2 or 3.

In preferred embodiments, none of H_(a) through H_(g) is a heterocyclicring.

In the above compounds, a pharmaceutically acceptable salt includes acidaddition salts and base addition salts.

Acid addition salts refer to those salts formed from compounds offormulae (1-4) and inorganic acids such as hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and/or organic acids such as acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like.

Base addition salts include those salts derived from compounds offormulae (1-4) and inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Suitable salts include the ammonium, potassium,sodium, calcium and magnesium salts derived from pharmaceuticallyacceptable organic non-toxic bases include salts of primary, secondary,and tertiary amines, substituted amines including naturally occurringsubstituted amines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-dimethylaminoethanol,2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine,arginine, histidine, caffeine, procaines, hydrabamine, choline, betaine,ethylenediamine, glucosamine, methylglucamine, theobromine, purines,piperazine, piperidine, N-ethylpiperidine, and the like.

In the above compounds and compositions, a hydrocarbyl group is formedexclusively from carbon and hydrogen, and includes, for example, any ofalkyl, alkenyl, alkynyl, aryl, aralkyl, alkylaryl, alkenyl-substitutedaryl, aryl-substituted alkenyl, alkynyl-substituted aryl,aryl-substituted alkynyl, biaryl, cycloalkyl, cycloalkenyl,bicycloalkyl, bicycloalkenyl, alkylcycloalkyl, alkenylcycloalkyl,alkynylcycloalkyl, aryl-substituted cycloalkyl, cycloalkyl-substitutedaryl, aryl-substituted cycloalkenyl, cycloalkenyl-substituted aryl,aryl-fused cycloalkyl and polycycloalkyl. A cyclohydrocarbyl group isalso formed exclusively from carbon and hydrogen, and includes at leastone ring, where exemplary cyclohydrocarbyl groups include any of aryl,aralkyl, alkylaryl, alkenyl-substituted aryl, aryl-substituted alkenyl,alkynyl-substituted aryl, aryl-substituted alkynyl, biaryl, cycloalkyl,cycloalkenyl, bicycloalkyl, bicycloalkenyl, alkylcycloalkyl,alkenylcycloalkyl, alkynylcycloalkyl, aryl-substituted cycloalkyl,cycloalkyl-substituted aryl, aryl-substituted cycloalkenyl,cycloalkenyl-substituted aryl, aryl-fused cycloalkyl and polycycloalkyl.In one aspect of the invention, the cyclohydrocarbyl group is selectedfrom cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. In anotheraspect, the cyclohydrocarbyl group is cyclopentyl.

A halocarbyl group is formed exclusively from carbon and halogen, andincludes the hydrocarbyl groups identified above wherein each hydrogenis replaced with a halogen selected from fluorine, chlorine, bromine andiodine, preferably fluorine and chlorine. A hydrohalocarbyl group, whichmay also be referred to as a halohydrocarbyl group, is formed fromexclusively from all of carbon, hydrogen and halogen, and includes thespecific hydrocarbyl groups identified above wherein some, but not all,of the hydrogen atoms are replaced with halogen atoms selected fromfluorine, chlorine, bromine and iodine, preferably fluorine and/orchlorine. Representative definitions of these hydrocarbyl groups (whichmay be substitued with halogen atoms to provide halocarbyl andhydrohalocarbyl derivatives thereof) are provided below.

“Alkyl” refers to an acyclic chain of carbon atoms which may be branchedor unbranched (linear). Methyl, ethyl, propyl (including n-propyl andiso-propyl) butyl (including n-butyl, iso-butyl, sec-butyl, andt-butyl), pentyl (including numerous isomers) and hexyl (includingnumerous isomers) are alkyl groups having 1 to 6 carbon atoms (commonlyreferred to as lower alkyl groups), and are exemplary of alkyl groups ofthe invention.

“Alkenyl” refers to an unsaturated aliphatic group having at least onedouble bond.

“Alkynyl” refers to an unsaturated hydrocarbon which may be eitherstraight- or branched-chain and have one or more triple bonds. Preferredgroups have no more than about 12 carbons atoms and may be ethyl,propynyl, 4-methylpentynl and so on, and structure isomers thereof.

“Aralkyl” refers to an alkyl group substituted by an aryl radical. Forexample, benzyl.

“Aralkynyl” refers to an alkynyl group substituted by an aryl ring. Forexample, ArC≡C—, ArCH₂CH₂CH₂C≡C— and so on.

“Cyloalkyl” refers to a cyclic arrangement of carbon atoms, wherecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl are cycloalkyl groupsof the invention having 3-6 carbon atoms. Additional groups within thescope of “cycloalkyl” as defined herein are polycycloalkyl groups,defined below.

“Cycloalkenyl” refers to a cyclic alkenyl group. Suitable cycloalkenylgroups include, for example, cyclopentenyl and cyclohexenyl.

A polycycloalkyl group is an arrangement of carbon atoms wherein atleast one carbon atom is a part of at least two separately identifiablerings. The polycycloalkyl group may contain bridging between two carbonatoms, where bicyclo[1.1.0]butyl, bicyclo[3.2.1]octyl,bicyclo[5.2.0]nonyl, tricycl[2.2.1.0¹]heptyl, norbornyl and pinanyl arerepresentative examples. The polycycloalkyl group may contain one ormore fused ring systems, where decalinyl (radical from decalin) andperhydroanthracenyl are representative examples. The polycycloalkylgroup may contain a spiro union, in which a single atom is the onlycommon member of two rings. Spiro[3.4]octyl, spiro[3.3]heptyl andspiro[4.5]decyl are representative examples.

“Halogen” refers to fluorine, chlorine, bromine and iodine.

As used herein, the following abbreviations have the indicated meanings:

Abbreviation Full name 5-ASA 5-aminosalicylic acid Ab Antibody ABTS2,2′-azino-di-[3-ethylbenzthiazoline sulphonate] ACD Acid citratedextrose AcOH Acetic Acid ACVP American College of Veterinary PracticeANOVA Analysis of Variance Ar Argon BCR-ABL Oncogene in chromosome 9:22translocation in CML BINAP 2,2′-Bis(diphenylphosphino)-1,1′-binaphthylBn Benzyl BnBr Benzyl Bromide BOC tert-Butoxycarbonyl cAMP Cyclicadenosine 3′-5′-monophosphate cat Catalytic CD Cluster designation CFAComplete Freund's adjuvant cGMP Cyclic guanosine 3′-5′-monophosphate CIACollagen Induced Arthritis CLL Chronic lymphocytic leukemia CML Chronicmyelogenous leukemia CNS Central Nervous System Con A Concanavalin A COXCyclooxygenase cPent Cyclopentyl cPentBr Cyclopentyl bromide CRE cAMPresponse element CsA Cyclosporin A DMAP 4-Dimethylaminopyridine DMARDDisease modifying anti-rheumatic drug DMF N,N-Dimethylformamide DMSOdimethylsulfoxide DNA Deoxyriboneucleic acid dppf1,1′-Bis(diphenylphosphino)ferrocene dppp1,3-Bis(diphenylphosphino)propane EC₅₀ Concentration at which a 50% ofmaximum observable effect is noted EDTA Ethylenediaminotetraacetic acidELISA Enzyme-linked immunosorbent assay EtOAc Ethyl acetate EtOH Ethylalcohol FBS Fetal bovine serum FCS Fetal calf serum fMLPFormyl-methionyl leucine phenylalanine g.i. Gastrointestinal H & EHaematoxylin and eosin HARBS High affinity rolipram binding site HBSSHanks Balanced Salt Solution HMPA Hexamethylphosphoramide HPLC Highpressure liquid chromatography i.p. intraperitoneal IBD Inflammatorybowel disease IBMX 3-isobutyl-1-methylxanthine IC Inhibitoryconcentration IC₅₀ Concentration at which 50% inhibition is observed IFAIncomplete Freund's adjuvant IFN-γ Interferon gamma IL Interleukin LAHLithium aluminum hydride LDA Lithium diisopropylamide LN Lymph node LPSlipopolysaccharide LTB4 Leukotriene B4 luc luciferase Me Methyl MeOHMethyl alcohol MHC Major histocompatibility class MLR Mixed lymphocytereaction MPO myeloperoxidase Ms Methanesulfonyl MsCl Methanesulfonylchloride NBS N-Bromosuccinimide n-BuLi n-Butyllithium n-BuSHn-Butanethiol NF-κB Nuclear factor kappa B NSAID Non-steroidalanti-inflammatory drug p.t. Post-transplant PBS Phosphate bufferedsaline Pcc Pigeon cytochrome C PDE Phosphodiesterase PEG Polyethyleneglycol PG Prostaglandin PMS Phenazine methosulfate PMSF Phenyl methylsulfonyl fluoride pTsOH p-Toluenesulfonic acid monohydrate Py PyridineRA Rheumatoid arthritis RF Rheumatoid factor R_(f) Retardation factorROS Reactive oxygen species RPMI Rosewell Park Memorial Institute RTXResiniferitoxin SAR Structure activity relationship TBAFTetrabutylammonium fluoride TBDMS tert-Butyldimethylsilyl TBDMSCItert-Butyldimethylsilyl chloride TCR T-cell receptor TEA TriethylamineTf Trifluoromethanesulfonyl TFA Trifluoroacetic acid Th T helper THFTetrahydrofuran TNBS Trinitrobenzene sulfonic acid TNF-α Tumour necrosisfactor alpha Trolox ® 6-hydroxy-2.5.7.8-tetramethylchroman-2-carboxylicacid TsOH p-Toluenesulfonic acid monohydrate XTT2,3-bis[2-methoxy-4-nitro-5-sulfo-phenyl]-2H- tetrazolium5-carboxanilide inner salt μM Micro molar

When any variable occurs more than one time in any constituent or incompounds of formulae (1-4), its definition on each occurrence isindependent of its definition at every other occurrence. Combinations ofsubstituents and/or variables are permissible only if such combinationsresult in stable compounds. The compounds useful in the methods andcompositions of the present invention, as well as the compounds of thepresent invention, may have asymmetric centers and occur as racemates,racemic mixtures and as individual diastereomers, or enantiomers withall isomeric forms being included in the present invention. A racemateor racemic mixture does not imply a 50:50 mixture of stereoisomers.

In another embodiment, the present invention provides pharmaceuticalcompositions containing a compound of formulae (1-4) as set forth above,in combination with a pharmaceutically-acceptable carrier, diluent orexcipient. These compositions may be used for the treatment inflammationor other conditions as disclosed herein. These compositions may also beformed into a medicament, which may used in the treatment of, forexample, inflammation.

These compositions are useful as, for example, assay standards,convenient means of making bulk shipments, or pharmaceuticalcompositions. An assayable amount of a compound of the invention is anamount which is readily measurable by standard assay procedures andtechniques as are well known and appreciated by those skilled in theart. Assayable amounts of a compound of the invention will generallyvary from about 0.001 wt % to about 80 wt % of the entire weight of thecomposition. Inert carriers include any material which does not degradeor otherwise covalently react with a compound of formulae (1-4).Examples of suitable inert carriers are water; aqueous buffers, such asthose which are generally useful in High Performance LiquidChromatography (HPLC) analysis; organic solvents, such as acetonitrile,ethyl acetate, hexane and the like; and pharmaceutically acceptablecarriers.

“Pharmaceutically acceptable carriers” for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). For example, sterile saline and phosphate-buffered salineat physiological pH may be used. Preservatives, stabilizers, dyes andeven flavoring agents may be provided in the pharmaceutical composition.For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. Id. at 1449. In addition,antioxidants and suspending agents may be used. Id.

Thus, the present invention provides a pharmaceutical or veterinarycomposition (hereinafter, simply referred to as a pharmaceuticalcomposition) containing a compound of formulae (1-4) as described above,in admixture with a pharmaceutically acceptable carrier. The inventionfurther provides a composition, preferably a pharmaceutical composition,containing an effective amount of a compound of (1-4) as describedabove, in association with a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention may be in anyform which allows for the composition to be administered to a patient.For example, the composition may be in the form of a solid, liquid orgas (aerosol). Typical routes of administration include, withoutlimitation, oral, topical, parenteral, sublingual, rectal, vaginal, andintranasal. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques. Pharmaceutical compositions of the invention areformulated so as to allow the active ingredients contained therein to bebioavailable upon administration of the composition to a patient.Compositions that will be administered to a patient take the form of oneor more dosage units, where for example, a tablet may be a single dosageunit, and a container of a compound of formulae (1-4) in aerosol formmay hold a plurality of dosage units.

Materials used in preparing the pharmaceutical compositions should bepharmaceutically pure and non-toxic in the amounts used. It will beevident to those of ordinary skill in the art that the optimal dosage ofthe active ingredient(s) in the pharmaceutical composition will dependon a variety of factors. Relevant factors include, without limitation,the type of subject (e.g., human), the particular form of the activeingredient, the manner of administration and the composition employed.

In general, the pharmaceutical composition includes an (where “a” and“an” refers here, and throughout this specification, as one or more)active compound of formulae (1-4) as described herein, in admixture withone or more carriers. The carrier(s) may be particulate, so that thecompositions are, for example, in tablet or powder form. The carrier(s)may be liquid, with the compositions being, for example, an oral syrupor injectable liquid. In addition, the carrier(s) may be gaseous, so asto provide an aerosol composition useful in, e.g., inhalatoryadministration.

When intended for oral administration, the composition is preferably ineither solid or liquid form, where semi-solid, semi-liquid, suspensionand gel forms are included within the forms considered herein as eithersolid or liquid.

As a solid composition for oral administration, the composition may beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like form. Such a solid composition willtypically contain one or more inert diluents or edible carriers. Inaddition, one or more of the following adjuvants may be present: binderssuch as carboxymethylcellulose, ethyl cellulose, microcrystallinecellulose, or gelatin; excipients such as starch, lactose or dextrins,disintegrating agents such as alginic acid, sodium alginate, Primogel,corn starch and the like; lubricants such as magnesium stearate orSterotex; glidants such as colloidal silicon dioxide; sweetening agentssuch as sucrose or saccharin, a flavoring agent such as peppermint,methyl salicylate or orange flavoring, and a coloring agent.

When the composition is in the form of a capsule, e.g., a gelatincapsule, it may contain, in addition to materials of the above type, aliquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition may be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid may be for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, preferred composition contain, inaddition to the present compounds, one or more of a sweetening agent,preservatives, dye/colorant and flavor enhancer. In a compositionintended to be administered by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the invention, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordigylcerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, cyclodextrin, propylene glycol or othersolvents; antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parenteral preparation can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid composition intended for either parenteral or oraladministration should contain an amount of a compound of formulae (1-4)such that a suitable dosage will be obtained. Typically, this amount isat least 0.01% of a compound of the invention in the composition. Whenintended for oral administration, this amount may be varied to bebetween 0.1% and about 70% of the weight of the composition. Preferredoral compositions contain between about 4% and about 50% of the activecompound of formulae (1-4). Preferred compositions and preparationsaccording to the present invention are prepared so that a parenteraldosage unit contains between 0.01% to 1% by weight of active compound.

The pharmaceutical composition may be intended for topicaladministration, in which case the carrier may suitably comprise asolution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, beeswax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device. Topical formulations may contain aconcentration of the compound of formulae (1-4) of from about 0.1% toabout 10% w/v (weight per unit volume).

The composition may be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release thedrug. The composition for rectal administration may contain anoleaginous base as a suitable nonirritating excipient. Such basesinclude, without limitation, lanolin, cocoa butter and polyethyleneglycol.

The composition may include various materials which modify the physicalform of a solid or liquid dosage unit. For example, the composition mayinclude materials that form a coating shell around the activeingredients. The materials which form the coating shell are typicallyinert, and may be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients may beencased in a gelatin capsule.

The composition in solid or liquid form may include an agent which bindsto the active component(s) and thereby assists in the delivery of theactive components. Suitable agents which may act in this capacityinclude a monoclonal or polyclonal antibody, a protein or a liposome.

The pharmaceutical composition of the present invention may consist ofgaseous dosage units, e.g., it may be in the form of an aerosol. Theterm aerosol is used to denote a variety of systems ranging from thoseof colloidal nature to systems consisting of pressurized packages.Delivery may be by a liquefied or compressed gas or by a suitable pumpsystem which dispenses the active ingredients. Aerosols of compounds ofthe invention may be delivered in single phase, bi-phasic, or tri-phasicsystems in order to deliver the active ingredient(s). Delivery of theaerosol includes the necessary container, activators, valves,subcontainers, spacers and the like, which together may form a kit.Preferred aerosols may be determined by one skilled in the art, withoutundue experimentation.

Whether in solid, liquid or gaseous form, the pharmaceutical compositionof the present invention may contain one or more known pharmacologicalagents used in the treatment of inflammation (including arthritis).

The pharmaceutical compositions may be prepared by methodology wellknown in the pharmaceutical art.

A composition intended to be administered by injection can be preparedby combining the compound of formulae (1-4) with water so as to form asolution. A surfactant may be added to facilitate the formation of ahomogeneous solution or suspension. Surfactants are compounds thatnon-covalently interact with the compound of formulae (1-4) so as tofacilitate dissolution or homogeneous suspension of the active compoundin the aqueous delivery system.

The compounds disclosed herein of formulae 1, 2, 3 or 4 (i.e., compoundsof formulae (1-4), or compounds of the present invention), orcompositions comprising one of more of these compounds and apharmaceutically acceptable carrier, diluent or excipient, may be usedin a method for treating or preventing an inflammatory condition ordisease in a patient, where the method comprises administering to thepatient in need thereof an amount of a compound or composition accordingto the present invention, where the amount is effective to treat orprevent the inflammatory condition or disease of the patient.

The inflammatory condition or disease may be an autoimmune condition ordisease; the inflammatory condition or disease may involve acute orchronic inflammation of bone and/or cartilage compartments of joints;the inflammatory condition or disease may be an arthritis selected fromrheumatoid arthritis, gouty arthritis or juvenile rheumatoid arthritis;the inflammatory condition or disease may be asthma; the condition ordisease may be associated with the disregulation of T-cells; thecondition or disease may be associated with elevated levels ofinflammatory cytokines (e.g., wherein the inflammatory cytokine is IL-2,or wherein the inflammatory cytokine is IFN-γ, or wherein theinflammatory cytokine is TNF-α); the inflammatory condition or diseasemay be multiple sclerosis; the inflammatory condition or disease may bepulmonary sarcadosis; the inflammatory condition or disease may beocular inflammation or allergy; the inflammatory condition or diseasemay be an inflammatory bowel disease (e.g., Crohn's disease orulcerative colitis); and the inflammatory condition or disease may be aninflammatory cutaneous disease (e.g., psoriasis or dermatitis).

Furthermore, the present invention provides a method for modulatingintracellular cyclic adenosine 5′-monophosphate levels within a patient,comprising administering to a patient in need thereof an amount of acompound or composition according to the present invention, wherein theamount is effective to modulate the intracellular cyclic adenosine5′-monophosphate levels of the patient. The patient may have aninflammatory condition or disease.

Furthermore, the present invention provides a method for treating orpreventing a disease or condition in a patient, where the disease orcondition is associated with pathological conditions that are modulatedby inhibiting enzymes associated with secondary cellular messengers, themethod comprising administering to a patient in need thereof an amountof a compound or a composition of the present invention, wherein theamount is effective to treat or prevent a disease or conditionassociated with pathological conditions that are modulated by inhibitingenzymes associated with secondary cellular messengers. The enzyme may bea cyclic AMP phosphodiesterase; or the enzyme may be a phosphodiesterase4; or the enzyme may be a phosphodiesterase 3; or the enzymes may beboth of phosphodiesterase 4 and phosphodiesterase 3; or the enzyme maybe a cyclic GMP phosphodiesterase.

Furthermore, the present invention provides a method of treating orpreventing transplant rejection in a patient, comprising administeringto a patient in need thereof an amount of a compound or composition ofthe present invention, where the amount is effective to treat or preventtransplant rejection in the patient. The rejection may be due to graftversus host disease.

Furthermore, the present invention provides a method of treating orpreventing uncontrolled cellular proliferation in a patient, comprisingadministering to a patient in need thereof an amount of a compound orcomposition according to the present invention, where the amount iseffective to treat or prevent uncontrolled cellular proliferation in thepatient. The uncontrolled cellular proliferation may be caused by acancer selected from leukemia and solid tumors.

Furthermore, the present invention provides a method of treating orpreventing conditions associated with the central nervous system (CNS)in a patient, comprising administering to a patient in need thereof anamount of a compound or composition according to the present invention,where the amount is effective to treat or prevent conditions associatedwith the central nervous system (CNS) in the patient. The conditionassociated with the central nervous system (CNS) may be depression.

In a method of the present invention, a compound of formulae (1-4), or acomposition comprising one or more compounds of formulae (1-4) and apharmaceutically acceptable carrier, diluent or excipient, may, althoughneed not, achieve one or more of the following desired results in thesubject to whom has been administered a compound of formulae (1-4) asdefined above, or a composition containing one of these compounds and apharmaceutically acceptable carrier, diluent or excipient:

1. Inhibition of reactive oxygen species generation from primaryneutrophils;

2. Inhibition of neutrophil chemotaxis;

3. Inhibition of TNF-α production;

4. Inhibition of edema;

5. Oxygen radical scavenging;

6. Inhibition of cyclic-AMP phosphodiesterases 1, 3 and/or 4, andrelated PDEs such as PDE7;

7. Potentiate induction of CRE-mediated transcription activity in humanmonocytic cells;

8. Inhibition of PDE, preferably PDE4, PDE3, or PDE3 and PDE4;

9. Inhibition of cytokine production by activated T-cell subsets;

10. Inhibition of neutrophil myeloperoxidase release;

11. Low ratio of IC₅₀PDE4(cat):IC₅₀PDE4(HARBS);

12. Inhibition of graft rejection;

13. Inhibition of clinical and histopathological parameters of diseasein inflammatory bowel disease; and

14. Inhibition of clinical and histopathological parameters of arthritisin a murine collage-induced arthritis model.

Thus, the inventive method may be used to treat inflammation, includingboth acute and chronic inflammation as well as certain proliferativedisorders (cancers). As used herein, inflammation includes, withoutlimitation, ankylosing spondylitis, arthritis (where this termencompasses over 100 kinds of rheumatic diseases), asthma, Crohn'sdisease, fibromyalgia syndrome, gout, inflammations of the brain(including multiple sclerosis, AIDS dementia, Lyme encephalopathy,herpes encephalitis, Creutzfeld-Jakob disease, and cerebraltoxoplasmosis), emphysema, inflammatory bowel disease, irritable bowelsyndrome, ischemia-reperfusion injury juvenile erythematosus pulmonarysarcoidosis, Kawasaki disease, osteoarthritis, pelvic inflammatorydisease, psoriatic arthritis (psoriasis), rheumatoid arthritis,psoriasis, tissue/organ transplant, scleroderma, spondyloarthropathies,systemic lupus erythematosus, pulmonary sarcoidosis, and ulcerativecolitis. As used herein, proliferative disorders includes, withoutlimitation, all leukemias and solid tumors that are susceptible toundergoing differentiation or apoptosis upon interruption of their cellcycle.

The inventive method provides for administering a therapeuticallyeffective amount of a compound of formulae (1-4), including salts,compositions etc. thereof. As used herein, the actual amount encompassedby the term “therapeutically effective amount” will depend on the routeof administration, the type of warm-blooded animal being treated, andthe physical characteristics of the specific warm-blooded animal underconsideration. These factors and their relationship to determining thisamount are well known to skilled practitioners in the medical arts. Thisamount and the method of administration can be tailored to achieveoptimal efficacy but will depend on such factors as weight, diet,concurrent medication and other factors which those skilled in themedical arts will recognize.

An effective amount of a compound or composition of the presentinvention will be sufficient to treat inflammation in a warm-bloodedanimal, such as a human. Methods of administering effective amounts ofanti-inflammatory agents are well known in the art and include theadministration of inhalation, oral or parenteral forms. Such dosageforms include, but are not limited to, parenteral solutions, tablets,capsules, sustained release implants and transdermal delivery systems;or inhalation dosage systems employing dry powder inhalers orpressurized multi-dose inhalation devices.

The dosage amount and frequency are selected to create an effectivelevel of the agent without harmful effects. It will generally range froma dosage of about 0.01 to 100 mg/Kg/day, and typically from about 0.1 to10 mg/Kg/day where administered orally or intravenously. Also, thedosage range will be typically from about 0.01 to 1 mg/Kg/day whereadministered intranasally or by inhalation.

The compounds of formulae (1-4) including the compounds used in themethods and compositions set forth above, may be prepared according tothe Schemes set forth in the following examples. The following examplesare offered by way of illustration and not by way of limitation.

Unless otherwise stated, flash chromatography and column chromatographymay be accomplished using Merck silica gel 60 (230-400 mesh). Flashchromatography may be carried out according to the procedure set forthin: “Purification of Laboratory Chemicals”, 3rd. edition,Butterworth-Heinemann Ltd., Oxford (1988), Eds. D. D. Perrin and W. L.F. Armarego, page 23. Column chromatography refers to the processwhereby the flow rate of eluent through a packing material is determinedby gravity. In all cases flash chromatography and radial chromatographymay be used interchangeably. Radial chromatography is performed usingsilica gel on a Chromatotron Model #7924T (Harrison Research, Palo Alto,Calif.). Unless otherwise stated, quoted R_(f) values are obtained bythin layer chromatography using Silica Gel 60 F₂₅₄ (Merck KGaA, 64271,Darmstadt, Germany).

Also, unless otherwise stated, chemical reactants and reagents wereobtained from standard chemical supply houses, such as Aldrich(Milwaukee, Wis.; www.aldrich.sial.com); EM Industries, Inc. (Hawthorne,N.Y.; www.emscience.com); Fisher Scientific Co. (Hampton, N.H.;www.fischer1.com); and Lancaster Synthesis, Inc. (Windham, N.H.;www.lancaster.co.uk). Gases were obtained from Praxair (Vancouver,B.C.). Cell lines, unless otherwise stated, where obtained from publicor commercial sources, e.g., American Tissue Culture Collection (ATCC,Rockville, Md.).

EXAMPLES

Compound 12, a representative compound of the invention, is preparedaccording to Schemes 1 and 2. Any number of compounds related tocompound 12 could be produced using similar methodology but startingwith different cinnamic acids. Compound 12 has the S-configuration atcarbon 3 (C3) which is directed by the configuration of the chiralauxiliary (which has the S-configuration) in compound 4. Alternativelythe R-configuration at C3 in compound 12 is generated using the chiralauxiliary with the opposite configuration (R). In the compoundsgenerated using methodology in Schemes 1 and 2, the products have anisomeric mixture at C5. Alternatively, compounds with fixedconfigurations at this center (C5) are generated according to Schemes 3and 4. Thus all of the diastereoisomers can be prepared using theappropriate methodology described in the following four Scheme s.

Focusing on compound 12, hydrogenation of the double bond in thecommercially available starting material para-hydroxy-meta-methoxycinnamic acid (1) is accomplished using H₂ in the presence of catalyst10% Pd/C. Protection of the para-hydroxy functionality in compound 2 isfollowed by the addition of the (S)-(−)-4-benzyl-2-oxazolidinone moietyto form compound 4. The use of this chiral auxiliary to direct thestereochemistry at positions α to carbonyls has been well documented.Alkylation of compound 4 with a substituted aryl bromide preparedaccording to Scheme 2 stereoselectively affords compound 5. Lithiumaluminum hydride reduction of compound 5 gives compound 6 containing theprimary alcohol. Protection of the primary alcohol as the silyl ether isfollowed by hydroboration of the double bond to afford compound 8.Benzylation then desilylation gives compound 10 which is oxidized usingJones' conditions to give the carboxylic acid 11. Hydrogenation inacetic acid affords the desired cyclized product 12.

Synthesis of Compound 2

A mixture of compound 1 (3.0 g, 0.0155 moles) and 10% Pd/C (150 mg) inAcOH/EtOAc (30 mL, 2:1) was stirred under H₂ (balloon) for 14 hours.After filtering through a celite plug, the solvent was evaporated underreduced pressure to provide compound 2 (2.98 g, 98%) as a white solidwhich was used without further purification.

Synthesis of Compound 3

Compound 2 (2.71 g, 13.8 mmol) was dissolved in dry DMF (25 mL) and thesolution was cooled to 0° C. NaH (2.21 g, 60% in mineral oil, 55.2 mmol)was added. After one hour, benzyl bromide (BnBr) (8.21 mL, 69.0 mmol)was added and the resulting mixture was stirred at room temperature foranother 16 hours. The mixture was diluted with diethyl ether (200 mL)and washed with saturated NaHCO₃ solution (2×75 mL) then H₂O (2×75 mL).The organic phase was dried with MgSO₄ and the solvent was evaporated todryness to afford the crude benzyl ether compound. This crude mixture,which contained an amount of the corresponding benzyl ester, wasdissolved in THF/MeOH/H₂O (50 mL, 2:1:1). LiOH.H₂O (1.74 g, 41.4 mmol)was added and the reaction mixture was stirred at room temperature for20 hours. After evaporation of the solvent to a small volume, themixture was extracted with CH₂Cl₂ (3×100 mL). The combined organic layerwas washed with H₂O (2×75 mL), dried over MgSO₄, filtered, and thefiltrate was evaporated to dryness. The residue was purified by columnchromatography on silica gel (hexanes/EtOAc, 2:1) to afford compound 3(3.2 g, 81%) as a white solid.

Synthesis of Compound 4

Solution 1: Triethylamine (2.9 mL, 21.0 mmol) followed by trimethylacetyl chloride (2.4 mL, 19.3 mmol) were added to a solution of compound3 (5.0 g, 17.5 mmol) in THF (40 mL) at 0° C. The mixture was stirred at0° C. for one hour.

Solution 2: In a second flask, n-butlyllithium (7.7 mL, 19.3 mmol) wasadded to a solution of (S)-(−)-4-benzyl-2-oxazolidinone (3.4 g, 19.3mmol) in dry THF (25 mL) at −78° C. This mixture was stirred for onehour and then added to the above anhydride (solution 1) via cannula. Themixture was warmed from 0° C. to room temperature then stirred for 24hours. The solution was diluted with saturated NaHCO₃ solution (150 mL),and extracted with CH₂Cl₂ (4×100 mL). The combined organic layer waswashed with H₂O (2×100 mL), dried over MgSO₄, filtered, and the filtratewas evaporated to dryness. The residue was purified by columnchromatography on silica gel (hexanes/EtOAc, 4:1) to afford compound 4(7.05 g, 91%) as a white crystalline solid.

Synthesis of Compound 5

Compound 4 (3.0 g, 6.73 mmol) was dissolved in dry THF (25 mL), cooledto −78° C. and 2.0 M LDA (in THF, 3.5 mL, 7.0 mmol) was added slowly.After one hour, a solution of compound 15 (3.2 g, 13.5 mmol) in THF (10mL) was added in one portion to the reaction solution, and the resultingmixture was warmed to 0° C. and stirred for an additional 2 hours. Theexcess base was quenched at 0° C. with saturated aqueous NaCl (100 mL),and the resulting solution was extracted with CH₂Cl₂ (3×100 mL). Thecombined organic layer was washed with saturated NaHCO₃ (2×100 mL), H₂O(2×100 mL), dried over MgSO₄, filtered, and the filtrate was evaporatedto dryness. The residue was purified by column chromatography on silicagel (hexanes/EtOAc, 3:1) to give compound 5 (2.63 g, 63%) as a colorlessoil.

Synthesis of Compound 6

Compound 5 (2.4 g, 3.86 mmol) in THF (5 mL) was added to a suspension ofLiAlH₄ (154.2 mg, 3.86 mmol) in THF (15 mL) at 0° C. The mixture wasstirred for 2 hours at 0° C. and quenched with saturated NaHCO₃ (1 mL)and 10% NaOH (1 mL). The resulting mixture was filtered, the filtratewas diluted with diethyl ether (150 mL), and washed with saturated NaCl(2×50 mL). The organic layer was dried over MgSO₄, filtered, and thefiltrate evaporated to dryness. The crude product was purified by flashcolumn chromatography, eluted with 2:1 hexanes/EtOAc to afford compound6 (1.47 g, 85%) as a colorless oil.

Synthesis of Compound 7

Compound 6 (1.50 g, 3.34 mmol) was stirred in dry CH₂Cl₂ (25 mL), thenEt₃N (0.56 mL, 4.01 mmol) was added followed by TBDMSCI (554.5 mg, 3.68mmol). The mixture was stirred at room temperature for 6 hours, dilutedwith EtOAc (200 mL) and washed with saturated NaHCO₃ solution (2×75 mL)and saturated NaCl solution (2×75 mL). The organic layer was dried overMgSO₄, filtered, and the filtrate evaporated to dryness. The residue waspurified by column chromatography on silica gel (hexanes/EtOAc, 5:1) togive compound 7 (1.74 g, 93%) as a colorless oil.

Synthesis of Compound 8

Compound 7 (560.0 mg, 0.995 mmol) was dissolved in THF (5 mL) and cooledto 0° C. BH₃-THF (1.0 M in THF, 1.0 mL, 1.0 mmol) was added dropwise.After the mixture was stirred at 0° C. for 5 hours, 10 N NaOH (1 mL) wasadded followed by 30% H₂O₂ (1 mL) and the mixture was stirred foranother 16 hours at room temperature. THF was evaporated, the mixturewas diluted with EtOAc (120 mL) and washed with saturated NaCl solution(2×30 mL). The organic layer was dried over MgSO₄, filtered and thefiltrate was evaporated to dryness. The residue was purified by columnchromatography on silica gel (hexanes/EtOAc, 2:1) to afford compound 8(435.5 mg, 75%) as a colorless oil.

Synthesis of Compound 9

Compound 8 (900.4 mg, 1.72 mmol) was dissolved in DMF (10 mL) and cooledto 0° C., and NaH (137.7 mg, 60% in mineral oil, 3.44 mmol) was added.After one hour, benzyl bromide (BnBr) (0.41 mL, 3.44 mmol) was added andthe resulting mixture was stirred at room temperature for another 16hours. The mixture was diluted with diethyl ether (150 mL) and washedwith H₂O (2×30 mL). The organic phase was then dried with MgSO₄,filtered and the filtrate was evaporated to dryness. The residue waspurified by column chromatography on silica gel (hexanes/EtOAc, 10:1) toafford compound 9 (915.1 mg, 88%) as a colorless oil.

Synthesis of Compound 10

Compound 9 (899.8 mg, 1.34 mmol) was dissolved in THF (10 mL), 1.0 Mtetrabutylammonium fluoride (in THF, 2.7 mL, 2.7 mmol) was added and theresulting mixture was stirred at room temperature for 8 hours. Thesolvent was evaporated, then the mixture was dissolved in EtOAc (100 mL)and washed with H₂O (2×40 mL). The organic phase was dried with MgSO₄,filtered and the filtrate was evaporated to dryness. The residue waspurified by column chromatography on silica gel (hexanes/EtOAc, 2:1) toafford compound 10 (731.6 mg, 98%) as a colorless oil.

Synthesis of Compound 11

Compound 10 (570.7 mg, 1.03 mmol) was dissolved in acetone (12 mL) andcooled to 0° C. Jones' reagent (aqueous 1N CrO₃ in 25% H₂SO₄, 2.06 mL,2.06 mmol) was added slowly over 5 minutes. The resulting dark greenmixture was stirred for 40 minutes at room temperature, diluted withEtOAc (150 mL) and washed with 5% HCl (2×40 mL) and H₂O (2×40 mL). Theorganic layer was then dried over MgSO₄, filtered, and the filtrate wasevaporated to give crude compound 11 (547.0 mg) which was used withoutfurther purification.

Synthesis of Compound 12

A mixture of compound 11 (547.0 mg, 0.946 moles) and 10% Pd/C (78.0 mg)in AcOH (10 mL) was stirred under H₂ (balloon) for 48 hours. Afterfiltering through a celite plug and evaporating the filtrate to dryness,the mixture was purified by reversed phase HPLC (column: Nova Pak HKC₁₈, M4063102 (Waters), 6μ, 19×300 mm) using 55% MeOH in water affordingcompound 12 (183.7 mg, 48% over two steps) as a colorless oil.

Synthesis of Compound 14

Potassium tert-butoxide (9.80 g, 0.0833 moles) was added to a suspensionof MePPh₃Br (29.8 g, 0.0833 moles) in dry toluene (180 mL) under argon.The mixture was stirred at room temperature for 2 hours.3′,4′-Dimethoxyacetophenone 13 (10.0 g, 0.0555 moles) was added as solidand the reaction mixture was stirred at room temperature for another 16hours. Water (10 mL) was added slowly and the mixture was diluted withEtOAc (200 mL), washed with saturated NaHCO₃ (2×200 mL), then with H₂O(2×200 mL). After drying over MgSO₄, the solution was filtered and thefiltrate was concentrated in vacuo. The residue was purified by columnchromatography on silica gel (hexanes/EtOAc, 19:1) to give compound 14(9.31 g, 93%) as a colorless oil.

Synthesis of Compound 15

N-bromosuccinimide (4.93 g, 27.38 mmol) and benzoyl peroxide (80.0 mg,0.330 mmol) were added to a solution of compound 14 (4.88 g, 27.38 mmol)in CHCl₃ (60 mL). The reaction mixture was stirred at room temperaturefor 30 minutes. The mixture was then diluted with EtOAc (200 mL), washedwith saturated sodium chloride (100 mL), dried (MgSO₄), filtered and thefiltrate was concentrated in vacuo. The resulting residue was purifiedusing silica gel column chromatography (hexanes/EtOAc, 97:3) to yieldcompound 15 (2.44 g, 37%) as a light yellow oil.

Compounds with defined stereochemistry at C5 can be synthesizedaccording to Scheme 3. Thus, the mixed anhydride, obtained fromcommercially available (3,4-dimethoxyphenyl) acetic acid 16 (Aldrich)and trimethylacetyl chloride, is reacted with the lithium anion of(S)-(−)-4-benzyl-2-oxazolidinone to afford compound 17. EnantioselectiveMichael addition of the titanium enolate of the chiral oxazolidinone 17to tert-butyl acrylate provided compound 18 having the carboxylatefunctionality with a suitable protecting group. Hydrolysis of the chiralauxiliary with lithium hydroxide and hydrogen peroxide yields thecarboxylic acid 19. Selective reduction of compound 19 with BH₃-THFgives compound 20 containing the primary alcohol. Removal of the t-butylester linkage with pTsOH.H₂O in toluene gives the corresponding hydroxylacid which is lactonized spontaneous to produce compound 21. Alkylationof the lithium anion of compound 21 with 4-(benzyloxy)-3-methoxybenzylbromide affords compound 22 as a mixture of diastereomers (˜1:1).Hydrogenation of compound 22 yields the desired product 23. Accordingly,any number of substituted benzyl bromides can be used to preparecompounds related to compound 23 with different substitution patternsabout the benzyl ring.

Synthesis of Compound 17

Solution 1: Triethylamine (12.8 mL, 91.7 mmol) followed by trimethylacetyl chloride (10.4 mL, 84.2 mmol) were added to a solution of(3,4-dimethoxyphenyl) acetic acid 16 (15.0 g, 76.5 mmol) in THF (120 mL)at 0° C. and the mixture was stirred for one hour.

Solution 2: In a second flask, n-bulyllithium (2.5 M in hexanes, 33.7mL, 84.2 mmol) was added to a solution of(S)-(−)-4-benzyl-2-oxazolidinone (14.9 g, 84.2 mmol) in dry THF (75 mL)at −78° C. This solution was stirred for one hour and then added tosolution 1 at 0° C. via cannula. The resultant mixture was warmed from0° C. to room temperature, stirred for 24 hours, then diluted withsaturated NaHCO₃ solution (300 mL), and extracted with CH₂Cl₂ (3×200mL). The combined organic layer was washed with saturated NaCl (2×150mL), dried over MgSO₄, filtered and the filtrate concentrated underreduced pressure. The residue was purified by column chromatography onsilica gel (hexanes/EtOAc, 4:1) to afford compound 17 (19.04 g, 70%) asa white solid.

Synthesis of Compound 19

Titanium isopropoxide (0.42 mL, 1.41 mmol) was added slowly into asolution of titanium tetrachloride (0.50 mL, 4.50 mmol) in drydichloromethane (10 mL) at 0° C. The resulting mixture was stirred at 0°C. for 5 minutes then diisopropylethylamine (1.04 mL, 5.91 mmol) wasadded. After 15 minutes, a solution of compound 17 (2.0 g, 5.63 mmol) indichloromethane (10 mL) was added. The mixture was stirred for 90minutes at 0° C., then tert-butyl acrylate (1.24 mL, 8.45 mmol) wasadded. The reaction mixture was warmed to room temperature and stirredfor 36 hours and then diluted with saturated ammonium chloride (50 mL).The aqueous layer was extracted with dichloromethane (3×50 mL) and thecombined organic layers were washed with 1 N HCl (2×75), water (2×75 mL)and saturated NaCl (2×75 mL). After drying over MgSO₄, filtration andevaporation of the filtrate in vacuo gave crude compound 18 (2.69 g)which was used without further purification.

Crude compound 18 (2.69 g) was dissolved in a mixture of 3:1 THF/water(85 mL) and cooled to 0° C. Lithium hydroxide monohydrate (466.6 mg,11.12 mmol) and 30% hydrogen peroxide (2.5 mL, 22.25 mmol) were addedand the mixture was stirred at 0° C. for 3 hours. A solution of sodiumsulfite (3.06 g, 24.46 mmol) was added followed by 0.5 N sodiumbicarbonate (41 mL). The mixture was stirred at room temperature for 2hours and then concentrated in vacuo. The aqueous phase was diluted with5% HCl to pH=2 and then extracted with EtOAc (3×75 mL). The combinedorganic layers were dried over MgSO₄, filtered and the filtrateconcentrated in vacuo. The crude product was purified by columnchromatography over silica gel using 0.2% acetic acid in 20% ethylacetate/hexanes to afford compound 19 (1.09 g, 60% over two steps) as alight yellow oil.

Synthesis of Compound 20

BH₃-THF (1.0 M solution in THF, 37.6 mL, 0.0376 moles) was addeddropwise over 20 minutes to a solution of compound 19 (12.18 g, 0.0376moles) in dry THF (50 mL) at −18° C. The cooling bath was then removed,and the reaction mixture was stirred at room temperature for 16 hours.Saturated NaHCO₃ solution (50 mL) was added, and the aqueous phase wasextracted with EtOAc (3×75 mL). The combined organic phase was washedwith saturated NaCl (2×75 mL). The organic phase was dried over MgSO₄,filtered and the filtrate evaporated in vacuo. The residue was purifiedby column chromatography, eluting with 50% EtOAc in hexanes to affordcompound 20 (10.52 g, 91%) as a colorless oil.

Synthesis of Compound 21

A solution of compound 20 (482.1 mg, 1.57 mmol) and p-toluenesulfonicacid monohydrate (44.9 mg, 0.236 mmol) in toluene (20 mL) was heated at80° C. for 30 minutes. The reaction mixture was diluted with EtOAc (100mL) and washed with saturated NaHCO₃ solution (2×40 mL). After dryingwith MgSO₄ , the mixture was filtered, and the filtrate concentrated togive compound 21 (342.1 mg, 92%) as a white solid.

Synthesis of Compound 22

Preparation of 4-(benzyloxy)-3-methoxybenzyl bromide: To a solution of4-(benzyloxy)-3-methoxybenzyl alcohol (9.0 g, 36.84 mmol) in anhydrousdiethyl ether (150 mL) was slowly added PBr₃ (4.99 g, 18.42 mmol) viasyringe, and the resulting mixture was stirred at room temperature for 3hours. The mixture was diluted with diethyl ether (100 mL) and washedwith saturated aqueous NaHCO₃ (2×75 mL) and brine (2×75 mL). The organiclayer was dried over anhydrous MgSO₄, and the solvent was removed underreduced pressure to afford 4-(benzyloxy)-3-methoxybenzyl bromide (10.9g, 96%) as a white solid.

n-Butyllithium (2.5 M solution in hexanes, 0.42 mL, 1.06 mmol) was addedto a solution of diisopropylamine (0.15 mL) in dry THF (10 mL) at −78°C. The mixture was stirred at −78° C. for 1 hour, then a solution ofcompound 21 (226.8 mg, 0.96 mmol) in THF (5 mL) was added. After 1 hour,a solution of 4-(benzyloxy)-3-methoxybenzyl bromide (248.5 mg, 0.80mmol, made according to literature procedures found in J. Org. Chem.1996, 61, 9146-9155) in THF (1 mL) was added in one portion to thereaction, and the resulting mixture was stirred at −78° C. for anadditional 4 hours. The excess base was quenched at 0° C. with saturatedaqueous NaCl (10 mL), and the resulting solution was extracted withEtOAc (3×20 mL). The combined organic layer was washed with saturatedNaCl (2×30 mL), dried over MgSO₄, filtered and the filtrate evaporatedto dryness. The residue was purified by column chromatography on silicagel (hexanes/EtOAc, 2:1) to give compound 22 (152.1 mg, 41%) as acolorless oil.

Synthesis of Compound 23

A mixture of compound 22 (150.0 mg, 0.324 mmol) and 10% Pd/C (22.5 mg)in EtOAc/AcOH (4:1, 5 mL) was stirred under H₂ (balloon) for 2 hours.The mixture was then filtered through a celite plug and the filtrate wasevaporated to dryness. The residue was purified by column chromatographyon silica gel (hexanes/EtOAc, 3:2) to give compound 23 (106.3 mg, 88%)as a colorless syrup.

Certain compounds of the present invention contain two asymmetric carbonatoms and thus there are four possible diastereomers for thesecompounds. An examplary synthetic sequence to prepare a specificstereoisomer is summarized in Scheme 4 below. Thus, protection of theprimary alcohol in compound 20 (Scheme 3) is accomplished using benzylbromide (BnBr) and sodium hydride in DMF to yield benzyloxy derivative24. Compound 24 is then converted to its corresponding acid 25 byreacting compound 24 with TFA. Synthesis of N-acyloxazolidinonederivative 26 from compound 25 is achieved using the same type of thereaction describe in previous sections. Stereoselective alkylation ofcompound 26 with 4-(benzyloxy)-3-methoxybenzyl bromide affords compound27. Hydrolysis of the chiral auxiliary with lithium hydroxide andhydrogen peroxide yields the carboxylic acid 28. Hydrogenation ofcompound 28 in acetic acid and lactonization with pTsOH.H₂O in toluenegives the desired cyclized product 29 which has the 3R, 5Sconfiguration. Accordingly, the three other diastereoisomers can besynthesized similarly but starting with different chiral oxazolidinones.For example, compound 30 which has the 3S, 5S configuration issynthesized using the intermediate analogous to compound 26 with theoxazolidinone containing the opposite (R) configuration.

Synthesis of Compound 24

Compound 20 (9.45 g, 30.45 mmol) was dissolved in DMF (100 mL) andcooled to 0° C., NaH (2.43 g, 60% in mineral oil, 60.90 mmol) was added.After one hour, BnBr (7.2 mL, 60.90 mmol) was slowly added and theresulting mixture was stirred at room temperature for another 16 hours.The mixture was diluted with diethyl ether (600 mL) and washed with H₂O(2×200 mL). The organic phase was dried over MgSO₄, filtered and thefiltrate was concentrated in vacuo. The residue was purified by columnchromatography on silica gel to afford compound 24 (10.73 g, 88%) as acolorless oil.

Synthesis of Compound 25

Trifluoroacetic acid (TFA) (25 mL) was added to a solution of compound24 (10.0 g, 29.00 mmol) in dichloromethane (100 mL) at room temperature.This mixture was stirred for 16 hours and then concentrated in vacuo.The resulting oil was purified using silica gel column chromatographyeluting with hexane-EtOAc-AcOH (75:23:2) to afford compound 25 (8.49 g,85%) as a colorless oil.

Synthesis of Compound 26

Solution 1: Triethylamine (0.98 mL, 6.82 mmol) followed by trimethylacetyl chloride (0.78 mL, 6.38 mmol) were added to a solution ofcompound 25 (2.00 g, 5.80 mmol) in THF (20 mL) at 0° C., and the mixturewas stirred for one hour.

Solution 2: In a second flask, n-bulyllithium (2.6 mL, 6.38 mmol) wasadded to a solution of (S)-(−)-4-benzyl-2-oxazolidinone (1.13 g, 6.38mmol) in dry THF (15 mL) at −78° C. This solution was stirred for onehour and then added to the above mixed anhydride (solution 1) viacannula. The resultant mixture at 0° C. was allowed to warm to roomtemperature. After stirring at room temperature for 24 hours, themixture was diluted with a saturated NaHCO₃ solution (150 mL), andextracted with CH₂Cl₂ (4×60 mL). The combined organic layer was washedwith H₂O (2×50 mL), dried over MgSO₄, filtered and the filtrateconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (hexanes/EtOAc, 4:1) to afford compound 26 (2.38 g, 81%)as a pale yellow oil.

Synthesis of Compound 27

n-BuLi (2.84 mL, 2.5 M solution in hexane, 7.10 mmol) was slowly addedto a solution of diisopropylamine (1.08 mL, 7.70 mmol) in dry THF (55mL) at −78° C. The reaction mixture was stirred at −78° C. under argonfor 1 hour at −78° C. Compound 26 in THF (35 mL) at −78° C. was addedand the reaction mixture was stirred for 1 hour. A solution of4-(benzyloxy)-3-methoxybenzyl bromide (2.73 g, 8.88 mmol) in THF (10 mL)was then added in one portion to the reaction. The resulting mixture waswarmed to 0° C. and stirred for an additional 2 hours. The excess basewas quenched at 0° C. with saturated aqueous NH₄Cl (100 mL), and theresulting solution was extracted with CH₂Cl₂ (3×100 mL). The combinedorganic layer was washed with saturated NaHCO₃ (2×50 mL), H₂O (2×50 mL),dried over MgSO₄, filtered and the filtrate was evaporated to dryness.The residue was purified by column chromatography on silica gel(hexanes/EtOAc, 3:1) to give compound 27 (3.30 g, 76%) as a white foam.

Synthesis of Compound 28

LiOH.H₂O (0.404 g, 9.60 mmol) and H₂O₂ (30% in H₂O, 2.2 mL, 19.20 mmol)were added to a solution of compound 27 (3.50 g, 4.80 mmol) in THF/H₂O(3:1, 67 mL) at 0° C. The reaction mixture was stirred at 0° C. for 3hours. A solution of Na₂SO₃ (2.66 g, 21.1 mmol, in water (30 mL)) wasthen added followed by a solution of 0.5 N NaHCO₃ (40 mL). The mixturewas stirred for 2 hours, and then the THF was evaporated in vacuo. Thisaqueous solution was diluted with 2N HCl to pH=2 and then extracted withEtOAc (3×250 mL). The combined organic layers were dried over MgSO₄,filtered and the filtrate was evaporated to dryness. The resulting oilwas purified using silica gel column chromatography eluting withhexane-EtOAc-AcOH (75:23:2) to afford compound 28 (2.23 g, 84%) as acolorless oil.

Synthesis of Compound 29

A mixture of compound 28 (1.86 g, 3.37 moles) and 10% Pd/C (180 mg) inAcOH (150 mL) was stirred under H₂ (balloon) 16 hours. The catalyst wasremoved by filtering the reaction mixture through a celite plug. Thefiltrate was evaporated to dryness and the residue was stirred withpTsOH.H₂O (200 mg) in toluene (100 mL) at 80° C. for 30 minutes. Thetoluene was removed in vacuo and the residue was purified by columnchromatography on silica gel (hexanes/EtOAc, 1:1) to afford compound 29(1.06 g, 85%) as a white foam.

Compounds with different alkoxy groups on the phenyl ring can besynthesized according to Scheme 5. For example, compound 43 can besynthesized as follows. Commercially available 3-hydroxy-4-methoxybenzylalcohol 31 is selectively protected as the benzyloxy derivative 32 bytreatment of 31 with benzyl bromide and potassium carbonate in refluxingtoluene to yield 89% of the desired product after crystallization.Compound 32 is then reacted with methanesulfonyl chloride in thepresence of triethylamine and CH₂Cl₂ to afford compound 33, which isused without further purification. The crude product 33 is then placedin DMF and treated with potassium cyanide in the presence of 18-crown-6.After work-up and purification, the nitrile 34 is isolated in 91% yieldover two steps. Hydrolysis of nitrile 34 with potassium hydroxide isthen achieved to afford the desire carboxylic acid 35 in 95% yield.Treatment of compound 35 with trimethylacetyl chloride gives a mixedanhydride which is reacted with the lithium anion of(S)-(−)-4-benzyl-2-oxazolidinone to furnish compound 36 in 75% yield.Enantioselective Michael addition of the titanium enolate of the chiraloxazolidinone 36 to tert-butyl acrylate provides compound 37 having thecarboxylate functionality with a suitable protecting group.Hydrogenation of compound 37 gave the alcohol in quantitative yield,which is converted to the cyclopentyloxy derivative 38 in 64% yield bytreatment with cyclopentyl bromide, potassium carbonate and potassiumiodide in DMF. Accordingly, any number of alkoxy derivatives on thephenyl ring can be made using the corresponding alkyl bromide orfunctionalized alkyl bromide. Hydrolysis of the chiral auxiliary withlithium hydroxide and hydrogen peroxide gives the carboxylic acid 39 in91% yield. Selective reduction of compound 39 with BH₃-THF affordscompound 40 (89% yield) containing the primary alcohol. The lactone 41is obtained in 94% yield by treatment of compound 40 with pTsOH.H₂O intoluene. Alkylation of compound 41 with 4-(benzyloxy)-3-methoxybenzylbromide affords compound 42 in 70% yield. Hydrogenation of compound 42in acetic acid gives the desired product 43 in 83% yield.

Synthesis of Compound 32

To a rapidly stirred slurry of 3-hydroxy-4-methoxybenzyl alcohol 31(30.0 g, 195 mmol), potassium carbonate (62.2 g, 450 mmol), and18-crown-6 (0.40 g, 1 mol %) in toluene (350 mL) was added a solution ofbenzyl bromide (25.6 g, 150 mmol) in toluene (150 mL) over 20 min. Thereaction mixture was refluxed for 16 hours, after which the mixture wasdiluted with diethyl ether (400 mL) and washed successively with NaOH (1N, 2×250 mL), saturated aqueous NaHCO₃ (2×250 mL), and brine (2×300 mL).The diethyl ether layer was dried over anhydrous MgSO₄, and the solventwas removed to provide a pale yellow solid (42.1 g) which wascrystallized with EtOAc and hexanes to give compound 32 (32.7 g, 89%) asa white crystalline solid.

Synthesis of Compound 33

Compound 32 (30.0 g, 122.8 mmol) was dissolved in dichloromethane (300mL) and cooled to 0° C., and then Et₃N (20.4 mL, 147.36 mmol) andmethanesulfonyl chloride (11.40 mL, 147.36 mmol) were added. The icebath was removed, and the solution was stirred at room temperature for 2hours. The mixture was then diluted with dichloromethane (700 mL),washed successively with saturated aqueous NaHCO₃ (2×300 mL) and H₂O(2×300 mL). The organic phase was dried over MgSO₄, filtered and thefiltrate was concentrated to afford compound 33 (33.08 g) as a paleyellow solid which was used for next step without further purification.

Synthesis of Compound 34

To a solution of crude 33 (33.08 g) in dry DMF (200 mL) were added KCN(15.99 g, 245.6 mmol) and 18-crown-6 (5.19 g, 19.65 mmol). The reactionmixture was stirred at room temperature for 18 hours, then poured intowater (1.5 L). The precipitate was collected and dissolved in EtOAc (600mL), washed with H₂O (2×200 mL) and brine (2×200 mL). The organic phasewas dried over MgSO₄, filtered and the filtrate was concentrated toafford compound 34 (28.5 g, 91% yield over two steps) as an off-whitesolid.

Synthesis of Compound 35

A mixture of compound 34 (28.0 g, 110.5 mmol) and KOH (94.0 g, 167.5mmol) in H₂O (170 mL) was heated at reflux for 12 hours. After thereaction mixture was cooled to room temperature, it was diluted with H₂O(1.6 L) and acidified with 12 N HCl to pH=2. The resulting precipitatewas collected and dried over P₂O₅ to give compound 35 (28.8 g, 95%) as awhite solid.

Synthesis of Compound 36

Solution 1: Triethylamine (17.7 mL, 126.92 mmol) followed by trimethylacetyl chloride (14.3 mL, 116.35 mmol) were added to a solution ofcompound 35 (28.8 g, 105.77 mmol) in THF (250 mL) at 0° C. and themixture was stirred for one hour.

Solution 2: In a second flask, n-bulyllithium (2.5 M in hexanes, 46.5mL, 116.35 mmol) was added to a solution of(S)-(−)-4-benzyl-2-oxazolidinone (20.6 g, 116.35 mmol) in dry THF (145mL) at −78° C. This solution was stirred for one hour and then added tosolution 1 at 0° C. The resultant mixture was warmed from 0° C. to roomtemperature, stirred for 24 hours, then diluted with saturated NaHCO₃solution (400 mL), and extracted with CH₂Cl₂ (4×300 mL). The combinedorganic layer was washed with brine (200 mL), dried over MgSO₄, filteredand the filtrate concentrated under reduced pressure. The residue waspurified by column chromatography on silica gel (hexanes/EtOAc, 4:1) toafford starting material (15.93 g) and compound 36 (15.30 g, 75% basedon recovery of starting material) as a white solid.

Synthesis of Compound 37

Titanium isopropoxide (2.6 mL, 8.69 mmol) was added slowly into asolution of titanium tetrachloride (3.1 mL, 27.8 mmol) in drydichloromethane (100 mL) at 0° C. The resulting mixture was stirred at0° C. for 5 minutes then diisopropylethylamine (6.7 mL, 38.24 mmol) wasadded. After 15 minutes, a solution of compound 36 (15 g, 34.76 mmol) indichloromethane (100 mL) was added. The mixture was stirred for 90minutes at 0° C., then tert-butyl acrylate (15.3 mL, 104.28 mmol) wasadded. The reaction mixture was stirred for 3 days at 0° C. and thendiluted with saturated ammonium chloride (300 mL). The aqueous layer wasextracted with dichloromethane (3×300 mL) and the combined organiclayers were washed with 5% HCl (2×400 mL), water (2×300 mL) andsaturated NaCl (400 mL). After drying over MgSO₄, filtration andevaporation of the filtrate in vacuo gave crude compound 37 (21.0 g). Aportion of crude 37 (2.1 g) was purified by silica gel columnchromatography eluted with EtOAc/Hexanes (1:2) to furnish the purecompound 37 (1.55 g) as a syrup.

Synthesis of Compound 38

A mixture of compound 37 (1.55 g, 2.77 mmol) and 10% Pd/C (150 mg) inEtOAc/AcOH (5:1, 60 mL) was stirred under H₂ (balloon) for 18 hours. Themixture was filtered on celite and the filtrate was evaporated todryness to provide the intermediate phenolic compound (1.30 g, 100%).

A suspension of the phenolic compound (0.30 g, 0.639 mmol), anhydrousK₂CO₃ (0.132 g, 0.958 mmol), and KI (5 mg) in dry DMF (1.5 mL) wasstirred and heated to 65° C., and then cyclopentyl bromide (0.10 mL,0.958 mmol) was added dropwise. The stirred mixture was heated at 65° C.for a further 21 hours. After cooling to room temperature, the reactionmixture was diluted with Et₂O (50 mL) and washed with H₂O (2×25 mL). Theorganic layer was dried with MgSO₄ and the solvent was evaporated. Theresidue was purified by silica gel column chromatography withhexanes/EtOAc (4:1) as eluent to yield compound 38 (0.22 g, 64%) as acolorless syrup.

Synthesis of Compound 39

Compound 38 (3.5 g, 6.51 mmol) was dissolved in THF/H₂O (3:1, 60 mL) andcooled to 0° C. Lithium hydroxide monohydrate (0.546 g, 13.02 mmol) and30% hydrogen peroxide (2.98 mL, 26.04 mmol) were added and the mixturewas stirred at 0° C. for 3 hours. A solution of sodium sulfite (3.61 g,28.64 mmol) in water (19 mL) was added, followed by 0.5 N sodiumbicarbonate (35 mL). The mixture was stirred at room temperature for 2hours and then concentrated in vacuo. The aqueous phase was diluted with5% HCl to pH=2 and then extracted with EtOAc (3×75 mL). The combinedorganic layers were dried over MgSO₄, filtered and the filtrateconcentrated in vacuo. The crude product was purified by columnchromatography over silica gel using 0.2% acetic acid in ethylacetate/hexanes (1:4) as eluent to afford compound 39 (2.24 g, 91%) as awhite solid.

Synthesis of Compound 40

BH₃-THF (1.0 M solution in THF, 2.70 mL, 2.70 mmol) was added dropwiseover 40 minutes to a solution of compound 39 (2.23 g, 2.64 mmol) in dryTHF (15 mL) at −18° C. The cooling bath was then removed, and thereaction mixture was stirred at room temperature for 18 hours. Saturatedaqueous NaHCO₃ solution (15 mL) was added, and the aqueous phase wasextracted with EtOAc (3×50 mL). The combined organic phase was washedwith saturated NaCl (2×50 mL). The organic phase was dried over MgSO₄,filtered and the filtrate evaporated in vacuo. The residue was purifiedby silica gel column chromatography, eluting with 25% EtOAc in hexanesto afford compound 40 (1.91 g, 89%) as a colorless oil.

Synthesis of Compound 41

A solution of compound 40 (0.39 g, 1.07 mmol) and p-toluenesulfonic acidmonohydrate (29 mg) in toluene (25 mL) was heated at 85° C. for 30minutes. The toluene was removed in vacuo, and the residue was dissolvedin dichloromethane (60 mL) and washed successively with saturatedaqueous NaHCO₃ (20 mL) and brine (20 mL). The organic phase was driedover MgSO₄, filtered and the filtrate was concentrated to give compound41 (0.293 g, 94%) as a white solid.

Synthesis of Compound 42

To a solution of compound 41 (0.29 g, 1.0 mmol) in dry THF (5 mL) underargon was slowly added LDA [1.20 mmol, prepared from n-BuLi (0.48 mL,2.5 M solution in hexane, 1.20 mmol) and diisopropylamine (0.17 mL, 1.20mmol)] in THF (2.5 mL) at −78° C. The mixture was stirred at −78° C. forone hour, and then HMPA (0.26 mL, 1.5 mmol) was added to the abovemixture via syringe. After 15 minutes, 4-(benzyloxy)-3-methoxybenzylbromide (0.614 g, 2.00 moles) in THF (1 mL) was added. The resultingmixture was slowly warmed to 0° C. and stirred for an additional 2hours. The excess base was quenched at 0° C. with saturated aqueousNH₄Cl (15 mL), and the resulting solution was extracted with CH₂Cl₂(3×50 mL). The combined organic layer was washed with brine (2×50 mL),dried over MgSO₄, filtered and the filtrate was evaporated to dryness.The residue was purified by column chromatography on silica gel(hexanes/EtOAc, 3:1) to give compound 42 (0.362 g, 70%) as a white foam.

Synthesis of Compound 43

A mixture of compound 42 (0.30 g, 0.581 mmol) and 10% Pd/C (30 mg) inEtOAc/AcOH (4:1, 10 mL) was stirred under H₂ (balloon) for 18 hours. Themixture was filtered on celite and the filtrate was evaporated todryness. The residue was purified by column chromatography on silica geleluted with hexanes/EtOAc (4:1) to afford compound 43 (0.205 g, 83%) asa white foam.

Synthesis of Lactam Compounds 51 and Related Analogues

δ-Lactams analogous to the δ-lactones can be synthesized according toScheme 6. For example, conversion of compound 20 into the keyintermediate 46 was achieved in a five steps sequence as follows.Treatment of compound 20 with zinc azide/bis-pyridine complex,triphenylphosphine and diisopropyl azodicarboxylate in toluene smoothlyaffords the corresponding azide 44 in 91% yield. Compound 44 is thenhydrogenated in the presence of 10% Pd—C and the resulting amine 45 isconverted into compound 46 (63% over two steps) by treatment with NaOHin DMF.

Towards the synthesis of compound 51, the first approach is depicted inScheme 7. In this approach, compound 46 is treated with NaH and benzylbromide in DMF to afford compound 47 in 80% yield. Compound 47 is thenplaced in THF and alkylated with 4-(benzyloxy)-3-methoxybenzyl bromideto give the desired coupling products 48 and 49 in a ratio of 7.2:1 in86% yield. These two isomers can be separated by silica gel columnchromatography. De-O-benzylation of compound 49 using 10% Pd/C ascatalyst proceeds readily and the phenol 50 is isolated in 98% yield.Further hydrogenolysis under high pressure can then afford compound 51.

Alternative methods to protect the lactam nitrogen can also be used asdepicted in Scheme 8. For example, N-protection of 46 as theN-t-butoxycarbonylamide can be achieved using di-tert-butyldicarbonateand triethylamine in dichloromethane to give derivative 52 in 95% yield.Alkylation of compound 52 with 4-(benzyloxy)-3-methoxybenzyl bromideaffords compound 53 in 67% yield. Compound 53 is a diasteromericmixture. Removal of the N-BOC protecting group in compound 53 withtrifluoroacetic acid in dichloromethane gives the product 54 in 74%yield. Hydrogenolysis of compound 54 using 10% Pd/C as catalyst providesthe diastereomeric mixture 55 in 81% yield.

Synthesis of Lactam Compounds 61 and 62

In another example of preparing lactams, the synthesis of compounds 61and 62 is achieved by the procedure depicted in Scheme 9. Treatment ofcompound 40 with zinc azide/bis-pyridine complex, triphenylphosphine anddiisopropyl azodicarboxylate in toluene affords the corresponding azide56. The crude compound 56 is then hydrogenated in the presence of 10%Pd—C to give compound 57 in 76% yield over two steps. The lactamcyclilization step involves a one-pot three-step reaction sequenceemploying tert-butyl ester solvolysis with p-toluenesulfonic acidmonohydrate, esterification in methanol, and lactam cyclization uponaddition of triethyl amine to provide compound 58 in a yield of 96% overthe three steps. N-protection of the resulting 58 with di-tert-butyldicarbonate and triethylamine in dichloromethane providesN-t-butoxycarbonylamide derivative 59 in 84% yield. Alkylation ofcompound 59 with LDA and 4-(benzyloxy)-3-methoxybenzyl bromide affordscompound 60 in 74% yield. Compound 60 is a diasteromeric mixture with aR/S ratio of 1:1. Removal of the N-BOC protecting group in compound 60with trifluoroacetic acid in dichloromethane gives the target product 61in 74% yield. Hydrogenolysis of compound 61 using 10% Pd/C as catalystprovides the final product 62 in 81% yield.

Synthesis of Compound 44

Preparation of ZnN₆.2Py complex: To a stirred solution of Zn(NO₃)₂.6H₂O(3.57 g, 12.0 mmol) in H₂O (6 mL) was added dropwise a solution of NaN₃(1.56 g, 24.0 mmol) in H₂O (12 mL). The white suspension was brought to50° C. in an oil bath, then pyridine (2.0 mL, 24.7 mmol) was addeddropwise forming a dense white precipitate. Stirring was continued whilethe mixture was slowly cooled to room temperature. The salt wasfiltered, washed with ice cold water and dried in vacuo to give ZnN₆.2Py(2.99 g, 81%) as a white solid.

Diisopropyl azodicarboxylate (1.30 mL, 6.59 mmol) was added to asuspension of compound 20 (1.0 g, 3.30 mmol), ZnN₆.2Py (0.76 g, 2.47mmol) and Ph₃P (1.73 g, 6.59 mmol) in anhydrous toluene (20 mL). Themixture was stirred at room temperature for 18 hours. The mixture wasconcentrated, and the residue was purified by column chromatography onsilica gel eluted with hexanes/EtOAc (9:1) to afford compound 44 (1.01g, 91%) as a colorless oil.

Synthesis of Compound 45

A mixture of compound 44 (1.00 g, 2.98 mmol) and 10% Pd/C (100 mg) inEtOAc (30 mL) was stirred under H₂ (balloon) for 18 hours. The mixturewas filtered on celite and the filtrate was evaporated to dryness togive compound 45 (0.923 g, 100%) as a colorless syrup.

Synthesis of Compound 46

Sodium hydroxide (5 N, 0.14 mL, 0.70 mmol) was added to a solution ofcompound 45 (0.21 g, 0.68 mmol) in THF (1 mL) and MeOH (1 mL). Themixture was stirred at room temperature for 18 hours. The mixture wasconcentrated, and the residue was purified by column chromatography onsilica gel eluted with hexanes/EtOAc (9:1) to afford compound 46 (0.091g, 63%) as a white solid.

Synthesis of Compound 47

Compound 46 (0.184 g, 0.782 mmol) was dissolved in DMF (5 mL) and cooledto 0° C., NaH (0.0344 g, 60% in mineral oil, 0.860 mmol) was added.After two hours, benzyl bromide (0.14 mL, 1.173 mmol) was slowly addedand the resulting mixture was stirred at room temperature for another 18hours. The solvent was evaporated and the resulting residue was purifiedby column chromatography on silica gel eluted with EtOAc to affordcompound 47 (0.203 g, 80%) as a white solid.

Synthesis of Compounds 48, 49

To a solution of compound 47 (0.23 g, 0.707 mmol) in dry THF (4 mL)under argon was slowly added LDA [0.85 mmol, prepared from n-BuLi (0.34mL, 2.5 M solution in hexane, 0.85 mmol) and diisopropylamine (0.12 mL,0.85 mmol)] in THF (2 mL) at −78° C. The mixture was stirred at −78° C.for one hour, and then HMPA (0.18 mL, 1.06 mmol) was added to the abovemixture via syringe. After 15 minutes, 4-(benzyloxy)-3-methoxybenzylbromide (0.434 g, 1.41 mmol) in THF (1 mL) was added. The resultingmixture was stirred for an additional 2 hours at −78° C. The excess basewas quenched at 0° C. with saturated aqueous NH₄Cl (10 mL), and theresulting solution was extracted with EtOAc (3×30 mL). The combinedorganic layers were washed with brine (2×40 mL), dried over MgSO₄,filtered and the filtrate was evaporated to dryness. The residue waspurified by column chromatography on silica gel eluted withhexanes/EtOAc (3:2) to give compounds 49 (0.295 g, 75.6%) and 48 (0.041mg, 10.5%) as white foams.

Synthesis of Compound 50

A mixture of compound 49 (0.25 g, 0.453 mmol) and 10% Pd/C (50 mg) inEtOAc (20 mL) was stirred under H₂ (balloon) for 48 hours. The mixturewas filtered on celite and the filtrate was evaporated to dryness togive compound 50 (0.204 g, 98%) as a white foam.

Synthesis of Compound 52

Di-tert-butyl dicarbonate (0.724 g, 3.32 mmol) was added to a solutionof compound 46 (0.39 g, 1.66 mmol), Et₃N (0.46 mL, 3.22 mmol) and DMAP(0.040 g) in CH₂Cl₂ (12 mL). The mixture was stirred at room temperaturefor 4 hours. The mixture was concentrated, and the residue was purifiedby column chromatography on silica gel eluted with hexanes/EtOAc (2:1)to afford compound 52 (0.543 g, 98%) as a white solid.

Synthesis of Compound 53

To a solution of compound 52 (0.54 g, 1.61 mmol) in dry THF (7 mL) underargon was slowly added LDA [1.93 mmol, prepared from n-BuLi (0.77 mL,2.5 M solution in hexane, 1.93 mmol) and diisopropylamine (0.27 mL, 1.93mmol)] in THF (4 mL) at −78° C. The mixture was stirred at −78° C. forone hour, and then HMPA (0.42 mL, 2.42 mmol) was added to the abovemixture via syringe. After 15 minutes, 4-(benzyloxy)-3-methoxybenzylbromide (0.989 g, 3.22 moles) in THF (2 mL) was added. The resultingmixture was stirred for an additional 4 hours at −78° C. The excess basewas quenched at 0° C. with saturated aqueous NH₄Cl (20 mL), and theresulting solution was extracted with EtOAc (4×50 mL). The combinedorganic layer was washed with saturated brine (2×50 mL), dried overMgSO₄, filtered and the filtrate was evaporated to dryness. The residuewas purified by column chromatography on silica gel eluted withhexanes/EtOAc (4:1) to give compound 53 (0.604 g, 67%) as a white foam.

Synthesis of Compound 54

Trifluoroacetic acid (10 mL) was added to a solution of compound 53(0.557 g, 0.990 mmol) in CH₂Cl₂ (10 mL). The mixture was stirred at roomtemperature for 2 hours and then concentrated in vacuo. The residue wasdissolved in CH₂Cl₂ (100 mL) and washed with saturated NaHCO₃ (3×20 mL).The organic layer was dried over MgSO₄, filtered and the filtrateconcentrated in vacuo. The crude product was purified by columnchromatography over silica gel using 5% MeOH in ethyl acetate as eluentto afford compound 54 (0.349 g, 76%) as a white foam.

Synthesis of Compound 55

A mixture of compound 54 (0.30 g, 0.65 mmol) and 10% Pd/C (30 mg) inEtOAc/AcOH (1:1, 10 mL) was stirred under H₂ (balloon) for 5 hours. Themixture was filtered on celite and the filtrate was evaporated todryness. The residue was purified by column chromatography on silica geleluted with EtOAc/MeOH (9:1) to afford compounds 55 (0.195 g, 81%) as awhite solid.

Synthesis of Compound 56

Diisopropyl azodicarboxylate (1.62 mL, 8.24 mmol) was added to asuspension of compound 40 (1.5 g, 4.12 mmol), ZnN₆.2Py (0.95 g, 3.09mmol) and Ph₃P (2.16 g, 8.24 mmol) in anhydrous toluene (20 mL). Themixture was stirred at room temperature for 18 hours. The mixture wasthen concentrated, and the residue was purified by column chromatographyon silica gel eluted with 15% of EtOAc in hexanes to afford compound 56(1.59 g) as a colorless oil.

Synthesis of Compound 57

A mixture of crude compound 56 (1.59 g) and 10% Pd/C (80 mg) in EtOAc(20 mL) was stirred under H₂ (balloon) for 20 hours. The mixture wasfiltered on celite and the filtrate was evaporated to dryness. Theresidue was purified by column chromatography on silica gel eluted withEtOAc/MeOH/Et₃N (85:14:1) to afford compound 57 (1.14 g, 76% over twosteps) as a colorless oil.

Synthesis of Compound 58

Compound 57 (0.301 g, 0.825 mmol) was dissolved in toluene (18 mL) andMeOH (2 mL) and treated with pTsOH.H₂O (0.472 g, 2.48 mmol). Thesolution was heated at reflux for 1.5 hours using a Dean-Starkapparatus. The Dean-Stark apparatus was then removed and Et₃N (0.35 mL,2.48 mmol) was added to the solution, which was heated at reflux for afurther 4 hours. The solvent was evaporated and the residue was purifiedby silica gel column chromatography eluted with 2% AcOH in EtOAc toprovide compound 58 (0.228 g, 96%) as a white solid.

Synthesis of Compound 59

Di-tert-butyl dicarbonate (0.935 g, 4.28 mmol) was added to a solutionof compound 58 (0.62 g, 2.14 mmol), Et₃N (0.60 mL, 4.28 mmol) and DMAP(0.060 g) in CH₂Cl₂ (20 mL). The mixture was stirred at room temperaturefor 5 hours. The mixture was concentrated, and the residue was purifiedby column chromatography on silica gel eluted with hexanes/EtOAc (3:1)to afford compound 59 (0.696 g, 84%) as a white solid.

Synthesis of Compound 60

To a solution of compound 59 (0.60 g, 1.54 mmol) in dry THF (5 mL) underargon was slowly added LDA [1.85 mmol, prepared from n-BuLi (0.74 mL,2.5 M solution in hexane, 1.85 mmol) and diisopropylamine (0.26 mL, 1.85mmol)] in THF (2 mL) at −78° C. The mixture was stirred at −78° C. forone hour, and then HMPA (0.40 mL, 2.30 mmol) was added to the abovemixture via syringe. After 15 minutes, 4-(benzyloxy)-3-methoxybenzylbromide (0.71 g, 2.30 mmol) in THF (2 mL) was added. The resultingmixture was stirred for an additional 4 hours at −78° C. The excess basewas quenched at 0° C. with saturated aqueous NH₄Cl (20 mL), and theresulting solution was extracted with EtOAc (3×60 mL). The combinedorganic layer was washed with saturated brine (2×50 mL), dried overMgSO₄, filtered and the filtrate was evaporated to dryness. The residuewas purified by column chromatography on silica gel eluted withhexanes/EtOAc (7:3) to give compound 60 (0.693 g, 74%) as a white foam.

Synthesis of Compound 61

Trifluoroacetic acid (3 mL) was added to a solution of compound 60 (0.63g, 1.02 mmol) in CH₂Cl₂ (3 mL). The mixture was stirred at roomtemperature for 4 hours, diluted with toluene (20 mL) and thenconcentrated in vacuo. The residue was purified by column chromatographyover silica gel using 5% MeOH in ethyl acetate as eluent to affordcompound 61 (0.39 g, 74%) as a white solid.

Synthesis of Compound 62

A mixture of compound 61 (0.28 g, 0.54 mmol) and 10% Pd/C (27 mg) inEtOAc/AcOH (1:1, 6 mL) was stirred under H₂ (balloon) for 5 hours. Themixture was filtered on celite and the filtrate was evaporated todryness. The residue was purified by column chromatography on silica geleluted with EtOAc/MeOH (97:3) to afford compound 62 (0.20 g, 84%) as awhite foam.

Preparation of Substituted Benzyl Bromides; Substituted Phenyl δ-LactoneIntermediates; and Final Products

Scheme 10 depicts general synthetic methodology that may be used for thesynthesis of δ-lactones C. Exemplary synthetic methodology to providecompounds A, B, and C is provided below.

Any number of substituted benzyl halides can be used to generate an endproduct of structure C with different substitution patterns on thebenzyl ring. Substituted benzyl bromides are available commercially ormay be generated from the corresponding substituted benzyl alcohol,benzaldehyde, benzoic acid or benzoic ester.

For example, substituted benzyl bromides can be prepared as outlined inScheme 11. Any number of compounds related to compound 63 could beproduced using similar methodology but starting with a differentsubstituted benzyl alcohol. Thus, treatment of commercially availablestarting material compound 31 with benzyl bromide and potassiumcarbonate in toluene gives the corresponding benzyloxy derivative 32,which is treated, without purification, with PBr₃ in diethyl ether togive desired bromide compound 63 in quantitative yield.

Synthesis of Compound 63

To a solution of alcohol 32 (3.20 g, 13.1 mmol) in anhydrous diethylether (15 mL) was added PBr₃ (1.77 g, 6.55 mmol) in one portion, and theresulting mixture was stirred at room temperature for 3 hours. Themixture was diluted with diethyl ether (40 mL) and washed with H₂O (2×30mL), saturated NaHCO₃ (2×30 mL), and brine (2×30 mL). The ether layerwas dried over anhydrous MgSO₄, and the solvent was removed underreduced pressure to afford compound 63 (4.02 g, 100%) as a light yellowsolid.

Substituted benzyl bromide compounds can also be prepared fromcommercially available substituted benzaldehydes, benzoic acids andbenzoic esters by first converting these compounds to the correspondingalcohol. Exemplary synthetic methodology to provide substituted benzylbromide from benzyl aldehyde is described below. For example, asillustrated in Scheme 12, treatment of 4-hydroxy-3-nitro-benzaldehyde 73with benzyl bromide in the presence of K₂CO₃ in DMF at 65° C. gives4-benzyloxy-3-nitro-benzaldehyde 74. Reduction of compound 74 inmethanol with NaBH₄ affords the alcohol 75 and subsequent brominationusing PBr₃ in diethyl ether provides the desired bromide compound 76.

Scheme 12 also shows how other representative benzyl bromide compoundsmay be made.

Synthesis of Compound 74

To a suspension of 4-hydroxy-3-nitrobenzaldehyde 73 (3.00 g, 17.95mmol), potassium carbonate (3.73 g, 26.93 mmol) in DMF (300 mL) wasslowly added benzyl bromide (2.85 mL, 23.96 mmol). The reaction mixturewas stirred at 65° C. for 18 hours. After cooling to room temperature,the mixture was diluted with water (140 mL) and extracted with diethylether (3×150 mL). The combined organic layers were washed with water(150 mL) and brine (150 mL). After drying over anhydrous MgSO₄,filtration and evaporation of the filtrate in vacuo gave crude compound74 (4.393 g, 95%) which was used for the next reaction without furtherpurification.

Synthesis of Compound 75

Compound 74 (4.30 g, 16.72 mmol) was dissolved in EtOH/CH₂Cl₂ (1:1, 50mL) and cooled to 0° C. NaBH₄ (0.63 g, 16.72 mmol) was addedportionwise. After the addition was completed, the ice-water bath wasremoved and the reaction mixture was stirred at room temperature for 2hours. Water (40 mL) was added and the mixture was extracted with CH₂Cl₂(3×60 mL). The combined organic layers were washed with brine (50 mL)and dried over anhydrous MgSO₄. Removal of the solvent gave a paleyellow solid which was purified by silica gel column chromatography(hexanes/EtOAc, 1:1) to give compound 75 (4.31 g, 99%) as a pale yellowsolid.

Synthesis of Compound 76

To a solution of compound 75 (4.30 g, 16.59 mmol) in anhydrous diethylether (40 mL) was slowly added PBr₃ (0.79 mL, 8.30 mmol) via syringe,and the resulting mixture was stirred at room temperature for 3 hours.The mixture was diluted with EtOAc (100 mL) and washed with saturatedaqueous NaHCO₃ (2×50 mL) and brine (2×50 mL). The organic layer wasdried over anhydrous MgSO₄, and the solvent was removed under reducedpressure to afford compound 76 (5.07 g, 95%) as a pale yellow solid.

Alkylation using various halide compounds to provide the desiredproducts 77, 80, 89, 92, 94, 95 and 96 is depicted in Schemes 13, 14,15, 16, 17, 18 and 19, respectively. In Scheme 13, compound 21 isalkylated with commercially available 3,4-difluorobenzyl bromide to givethe desired compound 77 in 59% yield.

Synthesis of Compound 77

n-Butyllithium (2.5 M solution in hexanes, 0.56 mL, 1.40 mmol) was addedto a solution of diisopropylamine (0.20 mL) in dry THF (10 mL) at −78°C. The mixture was stirred at −78° C. for 1 hour, then HMPA (0.33 mL,1.91 mmol) was added, followed by addition of a solution of compound 21(0.30 g, 1.27 mmol) in THF (3 mL). After 1 hour, a solution of3,4-difluoro benzyl bromide (purchased from Aldrich Chemical Company,Inc., 0.32 mL, 2.54 mmol)) was added in one portion to the reaction, andthe resulting mixture was stirred at −78° C. for an additional 4 hours.The excess base was quenched with saturated aqueous NH₄Cl (10 mL), andthe resulting solution was extracted with EtOAc (3×20 mL). The combinedorganic layer was washed with saturated NaCl (2×30 mL), dried overMgSO₄, filtered and the filtrate evaporated to dryness. The residue waspurified by column chromatography on silica gel (hexanes/EtOAc, 2:1) togive compound 77 (0.27 g, 59%) as a white solid.

In Scheme 14, compound 21 is alkylated with 3,4-dibenzyloxy benzylbromide (prepared by treatment the corresponding alcohol with PBr₃),followed by hydrogenation using 10% Pd/C as catalyst to give the desiredcompound 80 in good yield.

Synthesis of Compound 78

To a solution of 3,4-dibenzyloxybenzyl alcohol (1.35 g, 4.21 mmol) inanhydrous diethyl ether (25 mL) was added PBr₃ (0.20 mL, 2.11 mmol) inone portion, and the resulting mixture was stirred at room temperaturefor 3 hours. The mixture was diluted with diethyl ether (50 mL) andwashed with H₂O (2×30 mL), saturated NaHCO₃ (2×30 mL), and brine (2×30mL). The ether layer was dried over anhydrous MgSO₄, and the solvent wasremoved under reduced pressure to afford compound 78 (1.47 g, 91%) as alight yellow oil.

Synthesis of Compound 79

n-Butyllithium (2.5 M solution in hexanes, 0.38 mL, 0.931 mmol) wasadded to a solution of diisopropylamine (0.14 mL 0.999 mmol) in dry THF(3 mL) at −78° C. The mixture was stirred at −78° C. for 1 hour, thenHMPA (0.22 mL, 1.27 mmol) was added, followed by adding a solution ofcompound 21 (200.0 mg, 0.846 mmol) in THF (3 mL). After 1 hour, asolution of 3,4-dibenzyloxy benzyl bromide (compound 78, 248.5 mg, 0.80mmol) in THF (1 mL) was added in one portion to the reaction, and theresulting mixture was stirred at −78° C. for an additional 4 hours. Theexcess base was quenched with saturated aqueous NH₄Cl (10 mL), and theresulting solution was extracted with EtOAc (3×20 mL). The combinedorganic layer was washed with saturated NaCl (2×30 mL), dried overMgSO₄, filtered and the filtrate evaporated to dryness. The residue waspurified by column chromatography on silica gel (hexanes/EtOAc, 2:1) togive compound 79 (0.31 g, 67%) as a colorless oil.

Synthesis of Compound 80

A mixture of compound 79 (0.20 g, 0.37 mmol) and 10% Pd/C (25 mg) inEtOAc/AcOH (4:1, 5 mL) was stirred under H₂ (balloon) for 2 hours. Themixture was then filtered through a celite plug and the filtrate wasevaporated to dryness. The residue was purified by column chromatographyon silica gel (hexanes/EtOAc, 3:2) to give compound 80 (0.093 mg, 70%)as a colorless syrup.

Schemes 15 and 16 illustrate the preparation of compounds 89 and 92, tworepresentative compounds of the substituted phenyl 8-lactoneintermediates, using synthetic methodology similar to that described inprevious examples but using the appropriate benzyl alcohol to generatecompounds 89 and 92 (e.g., synthesis of compound 41 in Scheme 5).

Synthesis of Compound (90)

Lithium hydroxide monohydrate (3.21 g, 76.5 mmol) and H₂O₂ (30% in H₂O,17.5 mL, 152.8 mmol) were added a solution of compound 37 (21.4 g, 38.2mmol) in THF/H₂O (3:1, 350 mL) at 0° C. The reaction mixture was stirredat 0° C. for 3 hours. A solution of sodium sulfite (21.2 g, 168.1 mmol)in water (100 mL) was then added, followed by a solution of 0.5 N sodiumbicarbonate (200 mL). The mixture was stirred at room temperature for 2hours and then concentrated in vacuo. The aqueous phase was diluted with10% HCl to pH=2 and then extracted with EtOAc (3×700 mL). The combinedorganic layers were dried over MgSO₄, filtered and the filtrateconcentrated in vacuo. The resulting oil was purified using silica gelcolumn chromatography eluting with 0.2% acetic acid in ethylacetate/hexanes (1:3) to afford compound 90 (12.6 g, 82%) as a whitesolid.

Synthesis of Compound (91)

BH₃-THF (1.0 M solution in THF, 13.8 mL, 13.8 mmol) was added dropwiseover 20 minutes to a solution of compound 90 (5.53 g, 13.8 mmol) in dryTHF (40 mL) at −15° C. The cooling bath was then removed, and thereaction mixture was stirred at room temperature for 4 hours. Saturatedaqueous NaHCO₃ solution (20 mL) was added, and the aqueous phase wasextracted with EtOAc (3×20 mL). The combined organic phase was washedwith saturated NaCl (2×40 mL). The organic phase was dried over MgSO₄,filtered and the filtrate evaporated in vacuo. The residue was purifiedby silica gel column chromatography, eluting with EtOAc/hexanes (1:2) toafford compound 91 (5.22 g, 98%) as a colorless wax.

Synthesis of Compound (92)

A solution of Compound 91 (1.5 g, 3.88 mmol) and p-toluenesulfonic acidmonohydrate (86.0 mg) in toluene (30 mL) was heated at 80° C. for 30minutes. The toluene was removed in vacuo, and the residue was purifiedby silica gel column chromatography, eluting with EtOAc/hexanes (1:1) toafford compound 92 (1.11 g, 91%) as a colorless oil.

In Scheme 17, compound 41 is alkylated with compound 76 and subsequentcatalytic hydrogenation of the resulting compound 93 affords the desiredcompound 94 in good yield.

Synthesis of Compound 93

To a solution of compound 41 (0.20 g, 0.688 mmol) in dry THF (4 mL)under argon was slowly added LDA (1.16 mL, 0.826 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.20 mL, 1.03 mmol) wasadded to the mixture via syringe. After 15 minutes, compound 76 (0.332g, 1.03 mmol) was added. The resulting mixture was stirred at −78° C.for an additional 4 hours. Then reaction was quenched with saturatedaqueous NH₄Cl (10 mL), and the resulting solution was extracted withEtOAc (3×20 mL). The combined organic layers were washed with brine (30mL), dried over MgSO₄, and filtered, and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(benzene/EtOAc, 95:5) to give compound 93 (0.264 g, 72%) as a whitefoam.

Synthesis of Compound 94

A mixture of compound 93 (0.20 g, 0.376 mmol) and 10% Pd/C (30 mg) inEtOAc (7 mL) was stirred under H₂ (balloon) overnight. Catalyst wasremoved by filtration and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography (hexanes/EtOAc,7:3) to afford compound 94 (0.111 g, 72%) as a pale yellow solid. (Thesilica gel was pretreated with 1% triethylamine).

Similarly, in Scheme 18 and Scheme 19, compound 41 is alkylated withcommercially available methyl iodide and allyl iodide, respectively, toprovide the desired compound 95 and compound 96.

Synthesis of Compound 95

To a solution of compound 41 (0.20 g, 0.688 mmol) in dry THF (3 mL)under argon was slowly added LDA (1.16 mL, 0.826 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.2 mL, 1.03 mmol) wasadded to the above mixture via syringe. After 15 minutes, methyl iodide(0.064 mL, 1.03 mmol) was added. The resulting mixture was stirred at−78° C. for an additional 4 hours. Then reaction was quenched withsaturated aqueous NH₄Cl (5 mL), and the resulting solution was extractedwith EtOAc (3×15 mL). The combined organic layers were washed with brine(15 mL), dried over MgSO₄, and filtered, and the filtrate was evaporatedto dryness. The residue was purified by silica gel column chromatography(benzene/EtOAc, 95:5) to afford compound 95 (0.168 g, 80%) as a whitesolid.

Synthesis of Compound 96

To a solution of compound 41 (0.40 g, 1.38 mmol) in dry THF (4 mL) underargon was slowly added LDA (2.32 mL, 1.66 mmol, freshly prepared fromn-BuLi and diisopropylamine in THF at −78° C.). The mixture was stirredat −78° C. for one hour, and then HMPA (0.4 mL, 2.07 mmol) was added tothe mixture via syringe. After 15 minutes, allyl iodide (0.19 mL, 2.07mmol) was added. The resulting mixture was stirred at −78° C. for anadditional 4 hours. Then the reaction was quenched with saturatedaqueous NH₄Cl (10 mL), and the resulting solution was extracted withEtOAc (3×30 mL). The combined organic layers were washed with brine (30mL), dried over MgSO₄, and filtered, and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(benzene/EtOAc, 95:5) to afford compound 96 (0.33 g, 72%) as a whitesolid.

Compounds of formulae (1-4) with higher carbon number alkyloxy chainsattached to a phenyl ring, and particularly the phenyl ring at the 5position of the lactone or analog ring, have been prepared. For example,compound 101 can be produced in 5 steps from compound 91, as illustratedin Scheme 20. It should be recognized that the same or analogoussynthetic methodology can be applied to provide hydrocarbyloxysubstitution on a phenyl ring for any compound of or related tocompounds of formulae (1-4).

Thus, deprotection of compound 91 using H₂ and 10% palladium on carbongives the corresponding phenol derivative 97. Treatment of compound 97with 1-bromobutane, potassium carbonate and potassium iodide in DMFgives the butyloxy derivative 98. Removal of the t-butyl ester linkagein compound 98 with p-toluenesulfonic acid monohydrate in tolueneproduces the corresponding hydroxyl acid, which is lactonizedspontaneously to afford compound 99. Alkylation of the lithium anion ofcompound 99 with compound 76 yields compound 100. Hydrogenation ofcompound 100 provides the desired compound 101.

Synthesis of Compound 97

A mixture of compound 91 (3.0 g, 7.76 mmol) and 10% Pd/C (0.30 mg) inEtOAc/AcOH (1:1, 40 mL) was stirred under H₂ (balloon) overnight.Catalyst was removed by filtration and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(hexanes/EtOAc, 3:2) to afford compound 97 (1.82 g, 79%) as a whitesolid.

Synthesis of Compound 98

To a suspension of compound 97 (0.80 g, 2.70 mmol), potassium carbonate(0.746 g, 5.40 mmol) and KI (30 mg) in anhydrous DMF (7 mL) was added1-bromobutane (0.58 mL, 5.40 mmol) via syringe. Then the reactionmixture was stirred at 65° C. overnight. After cooling, the mixture wasdiluted with water (30 mL) and extracted with diethyl ether (3×50 mL).The combined organic layers were washed with brine (2×50 mL), dried overanhydrous MgSO₄, and concentrated to dryness. The residue was purifiedby silica gel column chromatography (hexanes/EtOAc, 7:3) to affordcompound 98 (0.834 g, 88%) as a white solid.

Synthesis of Compound 99

A mixture of compound 98 (0.834 g, 2.37 mmol) and p-toluenesulfonic acidmonohydrate (80.0 mg) in toluene (20.0 mL) was heated at 75° C. for 30minutes. Toluene was removed in vacuo, and the residue was purified bysilica gel column chromatography (hexanes/EtOAc, 7:3) to afford compound99 (0.422 g, 64%) as a colorless syrup.

Synthesis of Compound 100

To a solution of the lactone 99 (0.30 g, 1.08 mmol) in dry THF (4 mL)under argon was slowly added LDA (0.58 mL, 0.412 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.18 mL, 1.30 mmol) wasadded to the mixture via syringe. After 15 minutes, compound 76 (0.52 g,1.62 mmol) was added. The resulting mixture was stirred at −78° C. foran additional 4 hours. Then reaction was quenched with saturated aqueousNH₄Cl (10 mL), and the resulting solution was extracted with EtOAc (3×20mL). The combined organic layers were washed with brine (15 mL), driedover MgSO₄, and filtered, and the filtrate was evaporated to dryness.The residue was purified by silica gel column chromatography(benzene/EtOAc, 95:5) to afford compound 100 (0.443 g, 79%) as a lightyellow solid.

Synthesis of Compound 101

A mixture of compound 100 (0.40 g, 0.77 mmol) and 10% Pd/C (40 mg) inEtOAc (5 mL) was stirred under H₂ (balloon) overnight. Catalyst wasremoved by filtration and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography (benzene/EtOAc,3:2) to afford compound 101 (0.215 g, 70%) as a pale yellow foam.

Compounds of formulae (1-4) containing hydrocarbyloxycarbonylsubstitutions at a phenyl ring, and particularly the phenyl ring at C5of the lactone or analog ring, can be prepared according to Scheme 21.For example, compound 103 can be synthesized as follows. Lactonizationof compound 97 using p-toluenesulfonic acid monohydrate in toluene givescompound 102. Reaction of compound 102 with acetyl chloride andtriethylamine in dichloromethane provides compound 103 with the esterlinkage on phenyl ring.

Synthesis of Compound 102

A mixture of compound 97 (1.01 g, 3.41 mmol) and p-toluenesulfonic acidmonohydrate (100 mg) in toluene (30 mL) was heated at 75° C. for 30minutes. Toluene was removed in vacuo, and the residue was purified bysilica gel column chromatography (CH₂Cl₂/EtOAc, 70:30) to give compound102 (0.71 g, 94%) as a white solid.

Synthesis of Compound 103

To a solution of compound 102 (0.100 g, 0.45 mmol) in CH₂Cl₂ (2 mL) at0° C. were added triethylamine (0.125 mL, 0.9 mmol) and acetyl chloride(0.048 mL, 0.675 mmol). The reaction mixture was warmed to roomtemperature and stirred for 4 hours and then quenched with water (5 mL).The mixture was extracted with ethyl acetate (3×10 mL), and the combinedorganic layers were washed with brine (15 mL) and dried over MgSO₄.Solvent was removed and the resulting residue was purified by silica gelcolumn chromatography (benzene/EtOAc, 90:10) to give compound 103 (0.103g, 87%) as a colorless syrup.

Compounds of formulae (1-4) containing hydrocarbyl group substitution ona phenyl ring, and particularly the phenyl ring at position 5 of thelactone or analog ring, can be synthesized according to the reactionsequence depicted in Scheme 22. The same or analogous syntheticmethodology can be applied to provide hydrocarbon substitutions on aphenyl ring for any compound of, or compound related to, formulae (1-4).

As depicted in Scheme 22, protection of the hydroxyl group in compound91 is achieved using t-butyldimethylsilyl chloride and imidazole inN,N-dimethylformamide to give compound 104. Removal of the benzylprotecting group using hydrogen and palladium on carbon in ethyl acetateyields compound 105. Compound 105 is then converted to its correspondingaryl triflate 106 by reacting compound 105 with trifluoromethanesulfonicanhydride in pyridine. Palladium-catalyzed cross-coupling of compound106 with phenyl boronic acid affords compound 107. Lactonizationfollowed by alkylation furnishes compound 109. Finally, catalytichydrogenation provides compound 110.

Synthesis of Compound 104

Compound 91 (2.50 g, 6.50 mmol) was dissolved in dry DMF (20 mL), thenimidazole (0.664 g, 9.75 mmol) was added followed by TBDMSCl (1.47 g,9.75 mmol). The mixture was stirred at room temperature for 2 hours,diluted with diethyl ether (150 mL) and washed with saturated aqueousNaHCO₃ solution (2×50 mL) and water (2×50 mL). The organic layer wasdried over MgSO₄, filtered, and the filtrate was evaporated to drynessto give compound 104 (3.43 g) as a colorless oil which was used withoutfurther purification.

Synthesis of Compound 105

To a solution of compound 104 (3.43 g) in ethyl acetate (20 mL) wasadded 10% Pd on activated carbon (0.172 g). The flask was flushed withhydrogen and the mixture was stirred vigorously under an atmosphere ofhydrogen for a period of 16 hours. The mixture was filtered and thesolvent was evaporated. The residue was purified by silica gel columnchromatography (hexanes/ethyl acetate, 3:1) to afford compound 105 (2.72g, 85% over two steps) as a colorless oil.

Synthesis of Compound 106

To a solution of compound 105 (0.50 g, 1.22 mmol) in anhydrous pyridine(5 mL) at 0° C. was slowly added trifluoromethanesulfonic anhydride(0.23 mL, 1.34 mmol) via syringe, and the resulting mixture was stirredat 0° C. for 1 hour. The mixture was diluted with EtOAc (100 mL) andwashed with saturated aqueous brine (2×50 mL). The organic layer wasdried over anhydrous MgSO₄, and the solvent was removed under reducedpressure to afford compound 106 (0.623 g, 94%) as a pale yellow oil.

Synthesis of Compound 107

To a mixture of a solution of phenylboronic acid (0.0247 g, 0.202 mmol),compound 106 (0.10 g, 0.184 mmol), K₃PO₄ (0.0586 g, 0.276 mmol), and dry1,4-dioxane (2 mL) in a dry, Ar-flushed flask was added Pd(PPh₃)₄ (5.3mg, 0.00461 mmol). The mixture was stirred and heated in an 80° C. oilbath for a period of 16 hours. The cooled mixture was diluted with ethylacetate (75 mL) and washed with brine (2×25 mL). The organic layer wasthen dried with MgSO₄, filtered and the filtrate was evaporated todryness. The residue was purified by column chromatography on silica gel(hexanes/EtOAc, 30:1) to give compound 107 (0.0666 g, 87%) as acolorless oil.

Synthesis of Compound 108

A solution of compound 107 (0.060 g, 0.128 mmol) and p-toluenesulfonicacid monohydrate (12.2 mg, 0.064 mmol) in dry toluene (1 mL) was stirredunder an atmosphere of dry argon and heated to 80° C. for 2 hours. Thecooled solution was filtered through a column of flash silica gel andthe column was washed with hexanes/ethyl acetate (2:1, 150 mL).Evaporation of the solvent gave compound 108 (32.7 mg, 91%) as acolorless syrup.

Synthesis of Compound 109

To a solution of compound 108 in dry THF under argon was slowly addedLDA. The mixture was stirred at −78° C. for one hour, and then HMPA wasadded to the mixture via syringe. After 15 minutes, compound 76 wasadded. The resulting mixture was stirred at −78° C. for an additional 4hours. The reaction was quenched with saturated aqueous NH₄Cl and theresulting solution was extracted with EtOAc. The combined organic layerswere washed with brine, dried over MgSO₄, and filtered, and the filtratewas evaporated to dryness. The residue was purified by silica gel columnchromatography to afford compound 109.

Synthesis of Compound 110

A mixture of compound 109 and 10% Pd/C in EtOAc was stirred under H₂(balloon) overnight. Catalyst was removed by filtration and the filtratewas evaporated to dryness. The residue was purified by silica gel columnchromatography to give compound 110.

The synthesis of compound 114 is accomplished by the procedure depictedin Scheme 23. The synthetic methodology is similar to the synthesis ofcompound 110. Palladium-catalyzed cross-coupling of compound 106 withcommercially available B-benzyl-9-BBN (Aldrich Chemical Co., Milwaukee,Wis.) provides compound 111. Lactonization of compound 111 usingp-toluenesulfonic acid monohydrate in toluene affords compound 112.Akylation followed by catalytic hydrogenation gives the desired compound114.

Synthesis of Compound 111

To a mixture of a solution of B-benzyl-9-BBN (0.5 M, 1.32 mmol),compound 106 (0.650 g, 1.20 mmol), K₃PO₄ (0.382 g, 1.80 mmol), and dry1,4-dioxane (4 mL) in a dry, Ar-flushed flask was added(diphenylphosphino-ferrocene)palladium(II) chloride. The mixture wasstirred and heated in an 80° C. oil bath for a period of 18 hours. Thecooled mixture was diluted with ethyl acetate (20 mL), treated with 10%KOH (aq) and 30% H₂O₂ (aq, 1.0 mL each) and allowed to stir for 2 hours.The layers were separated and the organic phase was washed with brine(2×20 mL) before being dried (MgSO₄). The solution was filtered andevaporated to yield the crude product. This material was separated bycolumn chromatography (30:1 hexanes/ethyl acetate, 35 g of flash silicagel, 2.5 cm column). The fraction containing the spot at R_(f)=0.35(19:1 hexanes/ethyl acetate, uv 254 nm, phosphomolybdic acid, heat) wasisolated. Evaporation of the solvent gave compound 111 (0.295 g, 52%) asa colorless oil.

Synthesis of Compound 112

A solution of compound 111 (0.285 g, 0.603 mmol) and p-toluenesulfonicacid monohydrate (40.0 mg, 0.211 mmol) in dry toluene (5 mL) was stirredunder an atmosphere of dry Ar and heated to 80° C. for 3 hours. Thecooled solution was filtered though a column of flash silica gel (10 gin a 2 cm column) and the column was washed with hexanes/ethyl acetate(1:1, 150 mL). Evaporation of the solvent gave compound 112 (0.105 g,59%) as a colorless oil which slowly solidified on standing.

Synthesis of Compound 113

To a solution of diisopropylamine (56 μL, 0.40 mmol) in dry THF (6 mL)in a dry, Ar-flushed flask at −78° C. was added a solution oftert-butyllithium (1.7 M, 0.40 mmol). The solution was stirred for 15min and then compound 112 (0.100 g, 0.337 mmol) and dry THF (3 mL) wereadded by cannula. The reaction mixture was stirred for 1 h and thenbriefly warmed in an ice-water bath. Dry HMPA (88 μL, 0.51 mmol) wasadded and the solution was cooled to −78° C. Compound 76 (0.163 g, 0.506mmol) was added and the mixture was allowed to stir at −78° C. for aperiod of 3 h. The mixture was warmed to room temperature and thenopened to the air. The solvent was evaporated and the crude product wasimmediately purified by column chromatography (2:1 hexanes/ethylacetate, 20 g of flash silica gel, 2 cm column). The fraction containingthe spot at R_(f)=0.65 (1:1 hexanes/ethyl acetate, uv 254 nm,phosphomolybdic acid, heat) was isolated. Evaporation of the solventgave compound 113 (0.103 g, 57%) as a colorless oil.

Synthesis of Compound 114

To a solution of compound 113 (0.100 g, 0.186 mmol) in 4:1methanol/ethyl acetate (5 mL) was added 10% Pd on activated carbon (25mg, 0.023 mmol). The flask was flushed with hydrogen and the mixture wasstirred vigorously under an atmosphere of hydrogen for a period of 48hours. The mixture was filtered and the solvent was evaporated. Theresidue was purified by column chromatography (1:1 hexanes/ethylacetate, 15 g of flash silica gel, 2 cm column). The fraction containingthe spot at R_(f)=0.35 (1:1 hexanes/ethyl acetate, uv 254 nm,phosphomolybdic acid, heat) was isolated. Evaporation of the solventgave compound 114 (24.9 mg, 32%) as an off-white solid foam.

Compounds of formulae (1-4) may have boron substituent(s) on a phenylring, and particularly the phenyl ring that is attached at position 5 ofthe lactone or analog ring. Exemplary synthetic methodology to provideboron substitution is provided below. It should be recognized that thesame or analogous synthetic methodology can be applied to provide thesame or analogous substitution on a phenyl ring for any compound of, orrelated to, compounds of formulae (1-4).

For example, introduction of a boron functionality on a phenyl ring ofcompounds of formulae (1-4), and particularly on a phenyl ring atposition 5 of the lactone or analog of formulae (1-4), may beaccomplished as depicted in Scheme 24. Thus compound 115 could beproduced by palladium-catalyzed cross-coupling of compound 106 withbis(pinacolato)diboron using[1,1′-bis(diphenylphosphio)ferrocene]dichloropalladium(II) [PdCl₂(dppf)]as catalyst and 1-1′-bis(diphenylphosphio)ferrocene (dppf) as ligand.Lactonization of compound 115 using p-toluenesulfonic acid monohydratein toluene may afford compound 116. Alkylation of compound 116 withcompound 76 may provide compound 117. Catalytic hydrogenation ofcompound 117 using hydrogen and palladium on carbon may provide thedesired compound 118.

Compounds of formulae (1-4) may have nitrogen substitution(s) on aphenyl ring, and particularly a phenyl ring at position 5 of the lactoneor analog ring. Exemplary synthetic methodology to provide nitrogensubstitution is provided below. It should be recognized that the same oranalogous synthetic methodology can be applied to provide the same oranalogous substitution at a phenyl ring for any compound of, or relatedto, compounds of formulae (1-4).

For example, compound 122 could be prepared in a multi-step synthesisfrom compound 106, as depicted in Scheme 25. Thus, palladium-catalyzedamination of compound 106 using pyrrolidine, palladium acetate orpalladium dibenzylideneacetone, bis(diphenylphosphono)binaphthyl andsodium tert-butoxide in toluene may give compound 119. Lactonization ofcompound 119 using p-toluenesulfonic acid monohydrate in toluene mayafford compound 120. Alkylation of compound 120 with compound 76 mayprovide compound 121. Catalytic hydrogenation of compound 121 usinghydrogen and palladium on carbon may provide the desired compound 122.

Compounds of formulae (1-4) may have sulphur substitution(s) on a phenylring, and particularly the phenyl ring at position 5 of the lactone oranalog ring. Exemplary synthetic methodology to provide sulphursubstitutions at a phenyl ring is provided below. It should berecognized that the same or analogous synthetic methodology can beapplied to provide the same or analogous substitution at a phenyl ringof other compounds of formulae (1-4).

Placement of a sulphur-containing group on a phenyl ring may be achievedas depicted in Scheme 26.

Thus, an aryl triflate, such as compound 106, may be reacted with1-butanethiol, palladium acetate or palladium dibenzylideneacetone,bis(diphenylphosphono)binaphthyl and sodium tert-butoxide in toluene toprovide a compound, such as compound 123, with a carbon-sulphur bond.Lactonization may be achieved using p-toluenesulfonic acid monohydratein toluene to give the corresponding compound 124. Alkylation ofcompound 124 may then be accomplished by treatment of compound 124 withLDA and compound 76 in THF. Subsequent catalytic hydrogenation of theresulting compound 125 using hydrogen and palladium on carbon mayprovide the desired compound 126.

Compounds of formulae (1-4) may have phosphorus substitution(s) on aphenyl ring, and particularly the phenyl ring at carbon number 5 of thelactone or analog ring. Exemplary synthetic methodology to providephosphorus substitution is provided below. It should be recognized thatthe same or analogous synthetic methodology can be applied to providethe same or analogous substitution at a phenyl ring for any compound of,or related to, compounds of formulae (1-4).

Phosphorous substituent at the phenyl ring may be introduced accordingto the pathway depicted in Scheme 27 below. Thus, conversion of compound106 to compound 127 may be achieved by treatment of compound 106 withPdCl₂(PPh₃)₂, diethylphosphine, 1,3-bis(diphenylphosphino)propane (dppp)and diisopropylethylamine in DMF. Lactonization of compound 127 usingp-toluenesulfonic acid monohydrate in toluene may then provide thedesired compound 128.

As described in previous sections, compounds of the present inventioncontain two asymmetric carbon atoms and thus there are four possiblediastereomers for these compounds. Two exemplary synthetic sequences toprepare specific stereoisomers are depicted in Schemes 28 and 29,respectively, using methodology described in previous examples.

In Scheme 28, protection of the primary alcohol in compound 40 may beachieved using benzyl bromide (BnBr) and cesium carbonate in DMF toyield benzyloxy derivative 188, Compound 188 may then be converted toits corresponding acid 189 by reacting compound 188 with trifluoroaceticacid. Synthesis of N-acyloxazolidinone derivative 190 from compound 189may be accomplished using the same type of the reaction describe inprevious sections (for example, Scheme 4). Stereoselective alkylation ofcompound 190 with 4-(benzyloxy)-3-methoxybenzyl bromide may affordcompound 191. Hydrolysis of the chiral auxiliary with lithium hydroxideand hydrogen peroxide may yield the carboxylic acid 192. Hydrogenationof compound 192 in acetic acid and lactonization with p-toluenesulfonicacid monohydrate in toluene may give the desired cyclized product 193which has the 3R, 5S configuration.

Similarly, in Scheme 29, stereoselective alkylation of compound 26 with4-(cyclopentyloxy)-3-methoxybenzyl bromide (preparation of compound 199is described in Scheme 30) may afford compound 194. Hydrolysis of thechiral auxiliary with lithium hydroxide and hydrogen peroxide may yieldthe carboxylic acid 195. Hydrogenation of compound 195 in acetic acidand lactonization with p-toluenesulfonic acid monohydrate in toluene maygive the desired cyclized product 196 which has the 3R, 5Sconfiguration. Accordingly, the three other diastereoisomers related tocompound 193 or compound 196 can be synthesized similarly but startingwith different chiral oxazolidinones. For example, compounds containing3S, 5S configuration may be synthesized using the intermediate analogousto compound 26 or compound 190 with the oxazolidinone containing theopposite (R) configuration.

Scheme 30 and 31 illustrate the preparation of compound 199 and 203, twointermediates using to generate compounds 134, 136 and 194.

Thus, in Scheme 30, treatment of compound 197 with cyclopentyl bromide,potassium iodide and potassium carbonate in DMF gives the correspondingcyclopentyloxy derivative 198, which is treated with PBr₃ in diethylether to give desired bromide compound 199.

Synthesis of Compound 198

To a suspension of 4-hydroxy-3-methoxybenzyl alcohol 197 (1.00 g, 6.49mmol), potassium carbonate (1.79 g, 12.98 mmol) and potassium iodide(29.1 mg, 0.175 mmol) in DMF (10 mL) was slowly added cyclopentylbromide (0.91 mL, 8.44 mmol). The reaction mixture was stirred at 65° C.for 24 hours. After cooling to room temperature, the mixture was dilutedwith diethyl ether (50 mL) and washed with water (2×25 mL). After dryingover anhydrous MgSO₄, filtration and evaporation of the filtrate invacuo gave crude yellow, solid which was purified by silica gel columnchromatography (hexanes/EtOAc, 3:1) to give compound 198 (0.502 g, 35%)as a pale yellow solid.

Synthesis of Compound 199

To a solution of compound 198 (0.48 g, 2.17 mmol) in anhydrous diethylether (8 mL) was slowly added PBr₃ (0.10 mL, 1.09 mmol) via syringe, andthe resulting mixture was stirred at room temperature for 2 hours. Themixture was diluted with diethyl ether (50 mL) and washed with saturatedaqueous NaHCO₃ (2×25 mL) and brine (2×25 mL). The organic layer wasdried over anhydrous MgSO₄, and the solvent was removed under reducedpressure to afford compound 199 (0.577 g, 93%) as a white solid.

In Scheme 31, treatment of compound 200 with cyclopentyl bromide,potassium iodide and potassium carbonate in DMF gives the correspondingcyclopentyloxy derivative 201. Reduction of compound 201 with NaBH₄affords the alcohol 202 and subsequent bromination using PBr₃ in diethylether provides the desired bromide compound 203.

Synthesis of Compound 201

To a suspension of 3-hydroxy-4-methoxybenzaldehyde 200 (2.00 g, 13.2mmol), potassium carbonate (2.74 g, 19.8 mmol) and potassium iodide(60.0 mg, 0.361 mmol) in DMF (13 mL) was slowly added cyclopentylbromide (1.84 mL, 17.2 mmol). The reaction mixture was stirred at 65° C.for 21 hours. After cooling to room temperature, the mixture was dilutedwith toluene (100 mL), and the organic phase was washed with 1N NaOH(2×30 mL) and water (2×30 mL). After drying over anhydrous MgSO₄,filtration and evaporation of the filtrate in vacuo gave crude compound201 (2.70 g) which was used in the next step without furtherpurification.

Synthesis of Compound 202

Compound 201 (2.7 g) was dissolved in MeOH/CH₂Cl₂ (2:1, 15 mL) andcooled to 0° C. NaBH₄ (0.464 g, 12.26 mmol) was added portionwise. Thereaction mixture was stirred at 0° C. for 30 minutes. Water (100 mL) wasadded and the mixture was extracted with CH₂Cl₂ (3×30 mL). The combinedorganic layers were washed with brine (50 mL) and dried over anhydrousMgSO₄. Removal of the solvent gave a pale yellow solid which waspurified by silica gel column chromatography (hexanes/EtOAc, 1:1) togive compound 202 (2.65 g, 91% over two steps) as a colorless oil.

Synthesis of Compound 203

To a solution of compound 202 (0.50 g, 2.26 mmol) in anhydrous diethylether (9 mL) was slowly added PBr₃ (0.11 mL, 1.13 mmol) via syringe, andthe resulting mixture was stirred at room temperature for 2 hours. Themixture was diluted with diethyl ether (50 mL) and washed with saturatedaqueous NaHCO₃ (2×25 mL) and brine (2×25 mL). The organic layer wasdried over anhydrous MgSO₄, and the solvent was removed under reducedpressure to afford compound 203 (0.604 g, 94%) as a light yellow oil.

p-Substitution on Phenyl Ring

Compounds with different alkoxy groups substituted at the para positionon the phenyl ring may be synthesized according to Scheme 32 usingmethodology analogous to that described in previous sections. Forexample, compound 216 can be synthesized as follows. Commerciallyavailable 4-hydroxy-3-methoxybenzyl alcohol 204 (Aldrich, Milwaukee,Wis.) may be selectively protected as the benzyloxy derivative 205 bytreatment of 204 with benzyl bromide and potassium carbonate inrefluxing toluene. Compound 205 may then be reacted with methanesulfonylchloride in the presence of triethylamine and CH₂Cl₂ to afford compound206. Compound 206 may then be placed in DMF and treated with potassiumcyanide in the presence of 18-crown-6 to give the nitrile 207.Hydrolysis of nitrile 207 with 1 N aqueous potassium hydroxide may thenbe used to afford the desire carboxylic acid 208. Treatment of compound208 with trimethylacetyl chloride may give a mixed anhydride, which maybe reacted with the lithium anion of (S)-(−)-4-benzyl-2-oxazolidinone tofurnish compound 209. Enantioselective Michael addition of the titaniumenolate of the chiral oxazolidinone 209 to tert-butyl acrylate mayprovide compound 210 having the carboxylate functionality with asuitable protecting group. Hydrogenation of compound 210 may give thephenolic group, which may be protected as the cyclopentyloxy derivative211 by treatment with cyclopentyl bromide, potassium carbonate andpotassium iodide in DMF. Hydrolysis of the chiral auxiliary with lithiumhydroxide and hydrogen peroxide may give the carboxylic acid 212.Selective reduction of compound 212 with BH₃-THF may give compound 213containing the primary alcohol. The lactone 214 may be obtained bytreatment of compound 213 with pTsOH in toluene. Alkylation of compound214 with 4-(benzyloxy)-3-methoxybenzyl bromide may be used to affordcompound 215. Hydrogenation of compound 215 in acetic acid may be usedto give the desired product 216.

Scheme 33 illustrates the preparation of compound 218, using syntheticmethodology similar to that described in Scheme 16.

Compound 218 is a key intermediate which may be used to generatecompounds containing higher carbon number alkyloxy substitutions,hydrocarbyloxycarbonyl substitutions, hydrocarbyl substitutions, boronsubstitutions, nitrogen substitutions, sulphur substitutions andphosphorus substitutions at the para position of the phenyl ring usingthe synthetic methodology similar to that described in Schemes 20, 21,22, 23, 24, 25, 26, and 27 for functionalization of the meta position ofthe same phenyl ring.

Synthesis of Compound 142

n-Butyllithium (2.5 M solution in hexanes, 0.38 mL, 0.931 mmol) wasadded to a solution of diisopropylamine (0.14 mL, 0.999 mmol) in dry THF(3 mL) at −78° C. The mixture was stirred at −78° C. for 1 hour, thenHMPA (0.22 mL, 1.27 mmol) was added, followed by adding a solution ofcompound 21 (0.20 g, 0.846 mmol) in THF (3 mL). After 1 hour, a solutionof 3-benzyloxybenzyl bromide (0.469 g, 1.69 mmol) in THF (1 mL) wasadded in one portion to the reaction, and the resulting mixture wasstirred at −78° C. for an additional 4 hours. The excess base wasquenched with saturated aqueous NH₄Cl (10 mL), and the resultingsolution was extracted with EtOAc (3×20 mL). The combined organic layerwas washed with saturated NaCl (2×30 mL), dried over MgSO₄, filtered andthe filtrate evaporated to dryness. The residue was purified by columnchromatography on silica gel (hexanes/EtOAc, 2:1) to give compound 142(0.284 g, 78%) as a colorless oil.

Synthesis of Compound 131

A mixture of compound 142 (0.190 mg, 0.439 mmol) and 10% Pd/C (0.025 g)in EtOAc/AcOH (4:1, 5 mL) was stirred under H₂ (balloon) for 2 hours.The mixture was then filtered through a celite plug and the filtrate wasevaporated to dryness. The residue was purified by column chromatographyon silica gel (hexanes/EtOAc, 3:2) to give compound 131 (0.133 g, 89%)as a colorless syrup.

Synthesis of Compound 132

n-Butyllithium (2.5 M solution in hexanes, 0.38 mL, 0.931 mmol) wasadded to a solution of diisopropylamine (0.14 mL, 0.999 mmol) in dry THF(3 mL) at −78° C. The mixture was stirred at −78° C. for 1 hour, thenHMPA (0.22 mL, 1.27 mmol) was added, followed by adding a solution ofcompound 21 (0.20 g, 0.846 mmol) in THF (3 mL). After 1 hour, a solutionof 4-methoxybenzyl bromide (0.34 g, 1.69 mmol) in THF (1 mL) was addedin one portion to the reaction, and the resulting mixture was stirred at−78° C. for an additional 4 hours. The excess base was quenched withsaturated aqueous NH₄Cl (10 mL), and the resulting solution wasextracted with EtOAc (3×20 mL). The combined organic layer was washedwith saturated NaCl (2×30 mL), dried over MgSO₄, filtered and thefiltrate evaporated to dryness. The residue was purified by columnchromatography on silica gel (hexanes/EtOAc, 2:1) to give compound 132(0.221 g, 73%) as a colorless oil.

Synthesis of Compound 138

n-Butyllithium (2.5 M solution in hexanes, 0.38 mL, 0.931 mmol) wasadded to a solution of diisopropylamine (0.14 mL, 0.999 mmol) in dry THF(3 mL) at −78° C. The mixture was stirred at −78° C. for 1 hour, thenHMPA (0.22 mL, 1.27 mmol) was added, followed by adding a solution ofcompound 21 (0.20 g, 0.846 mmol) in THF (3 mL). After 1 hour, a solutionof 4-benzyloxybenzyl bromide (0.469 g, 1.692 mmol) in THF (1 mL) wasadded in one portion to the reaction, and the resulting mixture wasstirred at −78° C. for an additional 4 hours. The excess base wasquenched with saturated aqueous NH₄Cl (10 mL), and the resultingsolution was extracted with EtOAc (3×20 mL). The combined organic layerwas washed with saturated NaCl (2×30 mL), dried over MgSO₄, filtered andthe filtrate evaporated to dryness. The residue was purified by columnchromatography on silica gel (hexanes/EtOAc, 2:1) to give compound 138(0.29 g, 79%) as a colorless oil.

Synthesis of Compound 133

A mixture of compound 138 (190.0 mg, 0.324 mmol) and 10% Pd/C (25.0 mg)in EtOAc/AcOH (4:1, 5 mL) was stirred under H₂ (balloon) for 2 hours.The mixture was then filtered through a celite plug and the filtrate wasevaporated to dryness. The residue was purified by column chromatographyon silica gel (hexanes/EtOAc, 3:2) to give compound 133 (139.1 mg, 92%)as a colorless syrup.

Synthesis of Compound 134

n-Butyllithium (2.5 M solution in hexanes, 0.42 mL, 1.06 mmol) was addedto a solution of diisopropylamine (0.15 mL, 1.07 mmol) in dry THF (10mL) at −78° C. The mixture was stirred at −78° C. for 1 hour, then HMPA(0.22 mL, 1.27 mmol) was added, followed by adding a solution ofcompound 21 (226.8 mg, 0.96 mmol) in THF (5 mL). After 1 hour, asolution of 3-(cyclopentyloxy)-4-methoxybenzyl bromide (0.48 g, 1.68mmol) in THF (1 mL) was added in one portion to the reaction, and theresulting mixture was stirred at −78° C. for an additional 4 hours. Theexcess base was quenched with saturated aqueous NH₄Cl (10 mL), and theresulting solution was extracted with EtOAc (3×20 mL). The combinedorganic layer was washed with saturated NaCl (2×30 mL), dried overMgSO₄, filtered and the filtrate evaporated to dryness. The residue waspurified by column chromatography on silica gel (hexanes/EtOAc, 2:1) togive compound 134 (0.224 g, 60%) as a colorless oil.

Synthesis of Compound 135

n-Butyllithium (2.5 M solution in hexanes, 4.47 mL, 11.18 mmol) wasadded to a solution of diisopropylamine (1.57 mL, 11.18 mmol) in dry THF(28 mL) at −78° C. The mixture was stirred at −78° C. for one hour, thenHMPA (1.32 mL, 7.62 mmol) was added, followed by adding a solution ofcompound 21 (1.20 g, 5.08 mmol) in THF (27 mL). After 1 hour, a solutionof 3-(benzyloxy)-4-methoxybenzyl bromide (63) (3.12 g, 10.16 mmol) inTHF (5 mL) was added in one portion to the reaction, and the resultingmixture was stirred at −78° C. for an additional 4 hours. The excessbase was quenched with saturated aqueous NH₄Cl (100 mL), and theresulting solution was extracted with EtOAc (3×200 mL). The combinedorganic layer was washed with saturated NaCl (2×200 mL), dried overMgSO₄, filtered and the filtrate evaporated to dryness. The residue waspurified by column chromatography on silica gel (hexanes/EtOAc, 2:1) togive compound 135 (1.16 g, 73%) as a white foam.

Synthesis of Compound 136

n-Butyllithium (2.5 M solution in hexanes, 0.38 mL, 0.931 mmol) wasadded to a solution of diisopropylamine (0.14 mL, 0.999 mmol) in dry THF(3 mL) at −78° C. The mixture was stirred at −78° C. for one hour, thenHMPA (0.22 mL, 1.27 mmol) was added, followed by adding a solution ofcompound 21 (0.20 mg, 0.846 mmol) in THF (3 mL). After 1 hour, asolution of 4-(cyclopentyloxy)-3-methoxybenzyl bromide (199) (0.507 g,1.78 mmol) in THF (1 mL) was added in one portion to the reaction, andthe resulting mixture was stirred at −78° C. for an additional 4 hours.The excess base was quenched with saturated aqueous NH₄Cl (10 mL), andthe resulting solution was extracted with EtOAc (3×20 mL). The combinedorganic layer was washed with saturated NaCl (2×30 mL), dried overMgSO₄, filtered and the filtrate evaporated to dryness. The residue waspurified by column chromatography on silica gel (hexanes/EtOAc, 2:1) togive compound 136 (0.206 g, 55%) as a colorless oil.

Synthesis of Compound 137

Preparation of 4-(propyloxy)-3-methoxybenzyl bromide: To a suspension ofvanillin (2.00 g, 13.2 mmol), potassium carbonate (2.74 g, 19.8 mmol)and potassium iodide (60.0 mg, 0.361 mmol) in DMF (15 mL) was slowlyadded 1-bromopropane (1.56 mL, 17.2 mmol) via syringe. The reactionmixture was stirred at 65° C. for 5 hours. After cooling to roomtemperature, the mixture was diluted with diethyl ether (100 mL), andthe organic phase was washed with water (2×50 mL). After drying overanhydrous MgSO₄, filtration and evaporation of the filtrate in vacuogave crude 4-(propyloxy)-3-methoxybenzaldehyde (2.55 g) which was usedin the next step without further purification.

Crude 4-(propyloxy)-3-methoxybenzaldehyde (2.55 g) was dissolved in EtOH(30 mL) and cooled to 0° C. NaBH (0.499 g, 13.2 mmol) was addedportionwise. After the addition was completed, the ice-water bath wasremoved and the reaction mixture was stirred at room temperature for 2hours. Water (50 mL) was added and the resulting mixture was extractedwith diethyl ether (3×100 mL). The combined organic layers were driedover anhydrous MgSO₄. Removal of the solvent gave a pale yellow oilwhich was purified by silica gel column chromatography (hexanes/EtOAc,4:1) to give 4-(propyloxy)-3-methoxybenzyl alcohol (2.38 g, 92% over twosteps) as a colorless oil.

To a solution of 4-(propyloxy)-3-methoxybenzyl alcohol (2.28 g, 11.62mmol) in anhydrous diethyl ether (30 mL) was slowly added PBr₃ (0.55 mL,5.81 mmol) via syringe, and the resulting mixture was stirred at roomtemperature for 2 hours. The mixture was diluted with diethyl ether (150mL) and washed with saturated aqueous NaHCO₃ (2×75 mL) and brine (2×75mL). The organic layer was dried over anhydrous MgSO₄, and the solventwas removed under reduced pressure to afford4-(propyloxy)-3-methoxybenzyl bromide (2.94 g, 98%) as a white solid.

n-Butyllithium (2.5 M solution in hexanes, 0.56 mL, 1.40 mmol) was addedto a solution of diisopropylamine (0.20 mL, 1.42 mmol) in dry THF (4 mL)at −78° C. The mixture was stirred at −78° C. for one hour, then HMPA(0.33 mL, 1.91 mmol) was added, followed by adding a solution ofcompound 21 (0.30 g, 1.27 mmol) in THF (3 mL). After 1 hour, a solutionof 4-(propyloxy)-3-methoxybenzyl bromide (0.658 g, 2.54 mmol) in THF (3mL) was added in one portion to the reaction, and the resulting mixturewas stirred at −78° C. for an additional 4 hours. The excess base wasquenched with saturated aqueous NH₄Cl (15 mL), and the resultingsolution was extracted with EtOAc (3×25 mL). The combined organic layerwas washed with saturated NaCl (2×30 mL), dried over MgSO₄, filtered andthe filtrate evaporated to dryness. The residue was purified by columnchromatography on silica gel (hexanes/EtOAc, 2:1) to give compound 137(0.302 g, 57%) as a white foam.

Synthesis of Compound 139

n-Butyllithium (2.5 M solution in hexanes, 0.38 mL, 0.931 mmol) wasadded to a solution of diisopropylamine (0.14 mL, 0.999 mmol) in dry THF(3 mL) at −78° C. The mixture was stirred at −78° C. for 1 hour, thenHMPA (0.22 mL, 1.27 mmol) was added, followed by adding a solution ofcompound 21 (0.200 g, 0.846 mmol) in THF (3 mL). After 1 hour, asolution of benzyl bromide (0.10 mL, 0.846 mmol) was added in oneportion to the reaction, and the resulting mixture was stirred at −78°C. for an additional 4 hours. The excess base was quenched withsaturated aqueous NH₄Cl (10 mL), and the resulting solution wasextracted with EtOAc (3×20 mL). The combined organic layer was washedwith saturated NaCl (2×30 mL), dried over MgSO₄, filtered and thefiltrate evaporated to dryness. The residue was purified by columnchromatography on silica gel (hexanes/EtOAc, 2:1) to give compound 139(53 mg, 38%) as a colorless oil.

Synthesis of Compound 140

Preparation of 3-(propyloxy)-4-methoxybenzyl bromide: To a suspension of3-hydroxy-4-methoxybenzaldehyde (2.00 g, 13.2 mmol), potassium carbonate(2.74 g, 19.8 mmol) and potassium iodide (60.0 mg, 0.361 mmol) in DMF(15 mL) was slowly added 1-bromopropane (1.56 mL, 17.2 mmol) viasyringe. The reaction mixture was stirred at 65° C. for 5 hours. Aftercooling to room temperature, the mixture was diluted with diethyl ether(100 mL), and the organic phase was washed with water (2×50 mL). Afterdrying over anhydrous MgSO₄, filtration and evaporation of the filtratein vacuo gave crude 3-(propyloxy)-4-methoxybenzaldehyde (2.60 g) whichwas used in the next step without further purification.

Crude 3-(propyloxy)-4-methoxybenzaldehyde (2.60 g) was dissolved in EtOH(30 mL) and cooled to 0° C. NaBH₄ (0.499 g, 13.2 mmol) was addedportionwise. After the addition was completed, the ice-water bath wasremoved and the reaction mixture was stirred at room temperature for 2hours. Water (50 mL) was added and the resulting mixture was extractedwith diethyl ether (3×100 mL). The combined organic layers were driedover anhydrous MgSO₄. Removal of the solvent gave a pale yellow oilwhich was purified by silica gel column chromatography (hexanes/EtOAc,4:1) to give 3-(propyloxy)-4-methoxybenzyl alcohol (2.29 g, 89% over twosteps) as a colorless oil.

To a solution of 3-(propyloxy)-4-methoxybenzyl alcohol (2.28 g, 11.62mmol) in anhydrous diethyl ether (30 mL) was slowly added PBr₃ (0.55 mL,5.81 mmol) via syringe, and the resulting mixture was stirred at roomtemperature for 2 hours. The mixture was diluted with diethyl ether (150mL) and washed with saturated aqueous NaHCO₃ (2×75 mL) and brine (2×75mL). The organic layer was dried over anhydrous MgSO₄, and the solventwas removed under reduced pressure to afford3-(propyloxy)-4-methoxybenzyl bromide (2.92 g, 97%) as a white solid.

n-Butyllithium (2.5 M solution in hexanes, 0.56 mL, 1.40 mmol) was addedto a solution of diisopropylamine (0.20 mL, 1.42 mmol) in dry THF (4 mL)at −78° C. The mixture was stirred at −78° C. for one hour, then HMPA(0.33 mL, 1.91 mmol) was added, followed by adding a solution ofcompound 21 (0.30 g, 1.27 mmol) in THF (3 mL). After 1 hour, a solutionof 3-(propyloxy)-4-methoxybenzyl bromide (0.658 g, 2.54 mmol) in THF (3mL) was added in one portion to the reaction, and the resulting mixturewas stirred at −78° C. for an additional 4 hours. The excess base wasquenched with saturated aqueous NH₄Cl (15 mL), and the resultingsolution was extracted with EtOAc (3×25 mL). The combined organic layerwas washed with saturated NaCl (2×30 mL), dried over MgSO₄, filtered andthe filtrate evaporated to dryness. The residue was purified by columnchromatography on silica gel (hexanes/EtOAc, 2:1) to give compound 140(0.310 g, 59%) as a white foam.

Synthesis of Compound 141

n-Butyllithium (2.5 M solution in hexanes, 0.56 mL, 1.40 mmol) was addedto a solution of diisopropylamine (0.20 mL, 1.42 mmol) in dry THF (4 mL)at −78° C. The mixture was stirred at −78° C. for one hour, then HMPA(0.33 mL, 1.91 mmol) was added, followed by adding a solution ofcompound 21 (0.30 g, 1.27 mmol) in THF (3 mL). After 1 hour, a solutionof 4-fluoro benzyl bromide (0.32 mL, 2.54 mmol) was added in one portionto the reaction, and the resulting mixture was stirred at −78° C. for anadditional 4 hours. The excess base was quenched with saturated aqueousNH₄Cl (15 mL), and the resulting solution was extracted with EtOAc (3×25mL). The combined organic layer was washed with saturated NaCl (2×30mL), dried over MgSO₄, filtered and the filtrate evaporated to dryness.The residue was purified by column chromatography on silica gel(hexanes/EtOAc, 2:1) to give compound 141 (0.267 g, 61%) as a colorlesssyrup.

Synthesis of Compound 143

To a suspension of compound 97 (0.120 g, 0.41 mmol) and potassiumcarbonate (0.085 g, 0.615 mmol) in anhydrous DMF (2 mL) was added1-iodoethane (0.049 mL, 0.615 mmol) via syringe. The reaction mixturewas stirred at 65° C. overnight. After cooling, the mixture was dilutedwith water (10 mL) and extracted with diethyl ether (3×15 mL). Thecombined organic layers were washed with brine (10 mL), dried overanhydrous MgSO₄, and concentrated to dryness. The residue was purifiedby silica gel column chromatography (hexanes/EtOAc, 7:3) to affordcompound 143 (0.096 g, 72%) as a colorless syrup.

Synthesis of Compound 144

To a suspension of compound 97 (0.21 g, 0.71 mmol), potassium carbonate(0.147 g, 1.06 mmol) and KI (0.02 g) in anhydrous DMF (2 mL) was added1-bromopropane (0.077 mL, 0.85 mmol) via syringe. Then the reactionmixture was stirred at 65° C. overnight. After cooling, the mixture wasdiluted with water (10 mL) and extracted with diethyl ether (3×15 mL).The combined organic layers were washed with brine (10 mL), dried overanhydrous MgSO₄, and concentrated to dryness. The residue was purifiedby silica gel column chromatography (hexanes/EtOAc, 7:3) to affordcompound 144 (0.185 g, 77%) as a colorless syrup.

Synthesis of Compound 145

To a suspension of compound 97 (0.15 g, 0.51 mmol) and potassiumcarbonate (0.105 g, 0.76 mmol) in anhydrous DMF (2 mL) was added2-iodopropane (0.18 mL, 1.02 mmol) via syringe. Then the reactionmixture was stirred at 65° C. overnight. After cooling, the mixture wasdiluted with water (10 mL) and extracted with diethyl ether (3×15 mL).The combined organic layers were washed with brine (10 mL), dried overanhydrous MgSO₄, and concentrated to dryness. The residue was purifiedby silica gel column chromatography (hexanes/EtOAc, 7:3) to affordcompound 145 (0.18 g, 86%) as a colorless syrup.

Synthesis of Compound 146

To a suspension of compound 97 (0.15 g, 0.51 mmol) and potassiumcarbonate (0.105 g, 0.76 mmol in anhydrous DMF (2 mL) was added3-bromo-2-methylpropene (0.077 mL, 0.76 mmol) via syringe. Then thereaction mixture was stirred at 65° C. overnight. After cooling, themixture was diluted with water (10 mL) and extracted with diethyl ether(3×15 mL). The combined organic layers were washed with brine (10 mL),dried over anhydrous MgSO₄, and concentrated to dryness. The residue waspurified by silica gel column chromatography (hexanes/EtOAc, 7:3) toafford compound 146 (0.118 g, 66%) as a colorless syrup.

Synthesis of Compound 147

To a suspension of compound 97 (0.15 g, 0.51 mmol), potassium carbonate(0.105 g, 0.76 mmol) and KI (5.0 mg) in anhydrous DMF (2 mL) was added(bromomethyl)cyclobutane (0.085 mL, 0.76 mmol) via syringe. Then thereaction mixture was stirred at 65° C. overnight. After cooling, themixture was diluted with water (10 mL) and extracted with diethyl ether(3×15 mL). The combined organic layers were washed with brine (10 mL),dried over anhydrous MgSO₄, and concentrated to dryness. The residue waspurified by silica gel column chromatography (hexanes/EtOAc, 7:3) toafford compound 147 (0.132 g, 71%) as a white solid.

Synthesis of Compound 148

To a suspension of compound 97 (0.15 g, 0.51 mmol), potassium carbonate(0.105 g, 0.76 mmol) and KI (10 mg, cat.) in anhydrous DMF (2 mL) wasadded (bromomethyl)cyclohexane (0.077 mL, 0.76 mmol) via syringe. Thenthe reaction mixture was stirred at 65° C. overnight. After cooling, themixture was diluted with water (10 mL) and extracted with diethyl ether(3×15 mL). The combined organic layers were washed with brine (10 mL),dried over anhydrous MgSO₄, and concentrated to dryness. The residue waspurified by silica gel column chromatography (hexanes/EtOAc, 7:3) toafford compound 148 (0.124 g, 62%) as a colorless syrup.

Synthesis of Compound 149

To a suspension of compound 97 (0.15 g, 0.51 mmol) and potassiumcarbonate (0.105 g, 0.76 mmol) in anhydrous DMF (2 mL) was added1-iodopentane (0.099 mL, 0.76 mmol) via syringe. Then the reactionmixture was stirred at 65° C. overnight. After cooling, the mixture wasdiluted with water (10 mL) and extracted with diethyl ether (3×15 mL).The combined organic layers were washed with brine (10 mL), dried overanhydrous MgSO₄, and concentrated to dryness. The residue was purifiedby silica gel column chromatography (hexanes/EtOAc, 7:3) to affordcompound 149 (0.143 g, 77%) as a white solid.

Synthesis of Compound 150

To a suspension of compound 97 (0.15 g, 0.51 mmol) and potassiumcarbonate (0.105 g, 0.76 mmol) in anhydrous DMF (2 mL) was added1-iodohexane (0.11 mL, 0.76 mmol) via syringe. Then the reaction mixturewas stirred at 65° C. overnight. After cooling, the mixture was dilutedwith water (10 mL) and extracted with diethyl ether (3×15 mL). Thecombined organic layers were washed with brine (10 mL), dried overanhydrous MgSO₄, and concentrated to dryness. The residue was purifiedby silica gel column chromatography (hexanes/EtOAc, 7:3) to affordcompound 150 (0.148 g, 77%) as a white solid.

Synthesis of Compound 151

To a suspension of compound 97 (0.15 g, 0.51 mmol) and potassiumcarbonate (0.105 g, 0.76 mmol) in anhydrous DMF (2 mL) was added1-iodoheptane (0.13 mL, 0.76 mmol) via syringe. Then the reactionmixture was stirred at 65° C. overnight. After cooling, the mixture wasdiluted with water (10 mL) and extracted with diethyl ether (3×15 mL).The combined organic layers were washed with brine (10 mL), dried overanhydrous MgSO₄, and concentrated to dryness. The residue was purifiedby silica gel column chromatography (hexanes/EtOAc, 7:3) to affordcompound 151 (0.160 g, 80%) as a colorless syrup.

Synthesis of Compound 152

To a suspension of compound 97 (0.15 g, 0.51 mmol) and potassiumcarbonate (0.105 g, 0.76 mmol) in anhydrous DMF (2 mL) was added1-iodooctane (0.18 mL, 1.02 mmol) via syringe. Then the reaction mixturewas stirred at 65° C. overnight. After cooling, the mixture was dilutedwith water (10 mL) and extracted with diethyl ether (3×15 mL). Thecombined organic layers were washed with brine (10 mL), dried overanhydrous MgSO₄, and concentrated to dryness. The residue was purifiedby silica gel column chromatography (hexanes/EtOAc, 7:3) to affordcompound 152 (0.18 g, 86%) as a colorless syrup.

Synthesis of Compound 153

A mixture of compound 143 (0.096 g, 0.38 mmol) and p-toluenesulfonicacid monohydrate (10 mg, cat.) in toluene (5 mL) was heated at 75° C.for 30 minutes. Toluene was removed in vacuo, and the residue waspurified by silica gel column chromatography (hexanes/EtOAc, 65:35) togive compound 153 (0.060 g, 63%) as a white solid.

Synthesis of Compound 154

A mixture of compound 144 (0.179 g, 0.53 mmol) and p-toluenesulfonicacid monohydrate (20 mg, cat.) in toluene (5 mL) was heated at 75° C.for 30 minutes. Toluene was removed in vacuo, and the residue waspurified by silica gel column chromatography (hexanes/EtOAc, 65:35) togive compound 154 (0.086 g, 61%) as a colorless syrup.

Synthesis of Compound 155

A mixture of compound 145 (0.094 g, 0.278 mmol) and p-toluenesulfonicacid monohydrate (9 mg, cat.) in toluene (5 mL) was heated at 75° C. for30 minutes. Toluene was removed in vacuo, and the residue was purifiedby silica gel column chromatography (hexanes/EtOAc, 65:35) to givecompound 155 (0.069 g, 94%) as a colorless syrup.

Synthesis of Compound 156

A mixture of compound 146 (118 mg, 0.337 mmol) and p-toluenesulfonicacid monohydrate (10 mg, cat.) in toluene (5 mL) was heated at 75° C.for 30 minutes. Toluene was removed in vacuo, and the residue waspurified by silica gel column chromatography (hexanes/EtOAc, 65:35) togive compound 156 (0.092 g, 100%) as a colorless syrup.

Synthesis of Compound 157

A mixture of compound 147 (0.132 g, 0.362 mmol) and p-toluenesulfonicacid monohydrate (15 mg, cat.) in toluene (5 mL) was heated at 75° C.for 30 minutes. Toluene was removed in vacuo, and the residue waspurified by silica gel column chromatography (hexanes/EtOAc, 65:35) togive compound 157 (0.101 g, 96%) as a colorless syrup.

Synthesis of Compound 158

A mixture of compound 148 (0.124 g, 0.389 mmol) and p-toluenesulfonicacid monohydrate (15 mg, cat.) in toluene (5 mL) was heated at 75° C.for 30 minutes. Toluene was removed in vacuo, and the residue waspurified by silica gel column chromatography (hexanes/EtOAc, 65:35) togive compound 158 (0.09 g, 73%) as a white solid.

Synthesis of Compound 159

A mixture of compound 149 (0.143 g, 0.39 mmol) and p-toluenesulfonicacid monohydrate (20 mg, cat.) in toluene (5 mL) was heated at 75° C.for 30 minutes. Toluene was removed in vacuo, and the residue waspurified by silica gel column chromatography (hexanes/EtOAc, 65:35) togive compound 159 (0.112 g, 98%) as a colorless syrup.

Synthesis of Compound 160

A mixture of compound 150 (0.148 g, 0.389 mmol) and p-toluenesulfonicacid monohydrate (20 mg, cat.) in toluene (5 mL) was heated at 75° C.for 30 minutes. Toluene was removed in vacuo, and the residue waspurified by silica gel column chromatography (hexanes/EtOAc, 65:35) togive compound 160 (0.112 g, 94%) as a colorless syrup.

Synthesis of Compound 161

A mixture of compound 151 (0.148 g, 0.389 mmol) and p-toluenesulfonicacid monohydrate (30 mg, cat.) in toluene (5 mL) was heated at 75° C.for 30 minutes. Toluene was removed in vacuo, and the residue waspurified by silica gel column chromatography (hexanes/EtOAc, 65:35) togive compound 161 (0.12 g, 100%) as a colorless syrup.

Synthesis of Compound 162

A mixture of compound 152 (0.180 g, 0.44 mmol) and p-toluenesulfonicacid monohydrate (30 mg, cat.) in toluene (5 mL) was heated at 75° C.for 30 minutes. Toluene was removed in vacuo, and the residue waspurified by silica gel column chromatography (hexanes/EtOAc, 65:35) togive compound 162 (0.13 g, 88%) as a colorless syrup.

Synthesis of Compound 163

To a solution of compound 99 (0.11 g, 0.40 mmol) in dry THF (2 mL) underargon was slowly added LDA (0.67 mL, 0.48 mmol, freshly prepared fromn-BuLi and diisopropylamine in THF at −78° C.). The mixture was stirredat −78° C. for one hour, and then HMPA (0.1 mL, 0.60 mmol) was added tothe mixture via syringe. After 15 minutes, 4-(benzyloxy)-3-methoxybenzylbromide (0.184 g, 0.60 mmol) was added. The resulting mixture wasstirred at −78° C. for an additional 4 hours. The excess base wasquenched with saturated aqueous NH₄Cl (5 mL), and the resulting solutionwas extracted with EtOAc (3×15 mL). The combined organic layers werewashed with brine (15 mL), dried over MgSO₄, and filtered, and thefiltrate was evaporated to dryness. The residue was purified by silicagel column chromatography (benzene/EtOAc, 95:5) to afford compound 163(0.136 g, 67%) as a colorless syrup.

Synthesis of Compound 164

A mixture of compound 163 (0.136 g, 0.27 mmol) and 10% Pd/C (15 mg) inEtOAc (3 mL) was stirred under H₂ (balloon) overnight. The catalyst wasremoved by filtration and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography (bezene/EtOAc,19:1) to afford compound 164 (0.082 g, 73%) as a colorless syrup.

Synthesis of Compound 165

To a solution of compound 99 (0.30 g, 1.08 mmol) in dry THF (4 mL) underargon was slowly added LDA (1.8, mL, 1.3 mmol, freshly prepared fromn-BuLi and diisopropylamine in THF at −78° C.). The mixture was stirredat −78° C. for one hour, and then HMPA (0.28 mL, 1.62 mmol) was addedvia syringe. After 15 minutes, compound 76 (0.52 g, 1.62 mmol) wasadded. The resulting mixture was stirred at −78° C. for an additional 4hours. The excess base was quenched with saturated aqueous NH₄Cl (5 mL),and the resulting solution was extracted with EtOAc (3×15 mL). Thecombined organic layers were washed with brine (15 mL), dried overMgSO₄, and filtered, and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography (benzene/EtOAc,95:5) to afford compound 165 (0.443 g, 79%) as a bright yellow solid.

Synthesis of Compound 166

A mixture of compound 165 (0.136 mg, 0.27 mmol) and 10% Pd/C (15 mg) inEtOAc (3 mL) was stirred under H₂ (balloon) overnight. The catalyst wasremoved by filtration and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography (bezene/EtOAc,3:2) to afford compound 166 (0.215 g, 70%, R_(f)=0.29, benzene/EtOAc,9:4) as a pale yellow foam.

Synthesis of Compound 167

Preparation of (3,4-dibenzyloxy)benzyl bromide: To a solution of(3,4-dibenzyloxy)benzyl alcohol (1.35 g, 4.21 mmol) in anhydrous diethylether (25 mL) was added PBr₃ (0.20 mL, 2.11 mmol) in one portion, andthe resulting mixture was stirred at room temperature for 3 hours. Themixture was diluted with diethyl ether (50 mL) and washed with H₂O (2×30mL), saturated NaHCO₃ (2×30 mL), and brine (2×30 mL). The ether layerwas dried over anhydrous MgSO₄, and the solvent was removed underreduced pressure to afford (3,4-dibenzyloxy)benzyl bromide (1.47 g, 91%)as a white solid.

To a solution of compound 41 (0.10 g, 0.344 mmol) in dry THF (2 mL)under argon was slowly added LDA (0.53 mL, 0.379 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.09 mL, 0.517 mmol) wasadded via syringe. After 15 minutes, (3,4-dibenzyloxy)benzyl bromide(0.198 g, 0.517 mmol) was added. The resulting mixture was stirred at−78° C. for two hours and then allowed to warm up to room temperatureover a period of two hours. The excess base was quenched with saturatedaqueous NH₄Cl (5 mL), and the resulting solution was extracted withEtOAc (3×15 mL). The combined organic layers were washed with brine (15mL) dried over MgSO₄, and filtered, and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(benzene/EtOAc, 95:5) to afford compound 167 (0.061 g, 30%) as acolorless syrup.

Synthesis of Compound 168

To a solution of compound 41 (0.10 g, 0.344 mmol) in dry THF (2 mL)under argon was slowly added LDA (0.53 mL, 0.379 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.09 mL, 0.517 mmol) wasadded via syringe. After 15 minutes, benzyl bromide (0.062 mL, 0.517mmol) was added. The resulting mixture was stirred at −78° C. for twohours and then allowed to warm up to room temperature over a period oftwo hours. The excess base was quenched with saturated aqueous NH₄Cl (5mL), and the resulting solution was extracted with EtOAc (3×15 mL). Thecombined organic layers were washed with brine (15 mL), dried overMgSO₄, and filtered, and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography (benzene/EtOAc,95:5) to afford compound 168 (0.051 g, 39%) as a colorless syrup.

Synthesis of Compound 169

To a solution of compound 41 (0.10 g, 0.344 mmol) in dry THF (2 mL)under argon was slowly added LDA (0.53 mL, 0.379 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.09 mL, 0.517 mmol) wasadded via syringe. After 15 minutes, commercially available(3-trifluoromethyl)benzyl bromide (Aldrich Chemical Co., Milwaukee,Wis., 0.079 mL, 0.517 mmol) was added. The resulting mixture was stirredat −78° C. for an additional 4 hours. The excess base was quenched withsaturated aqueous NH₄Cl (5 mL), and the resulting solution was extractedwith EtOAc (3×15 mL). The combined organic layers were washed with brine(15 mL), dried over MgSO₄, and filtered, and the filtrate was evaporatedto dryness. The residue was purified by silica gel column chromatography(benzene/EtOAc, 95:5) to afford compound 169 (0.126 g, 82%) as acolorless syrup.

Synthesis of Compound 170

To a solution of compound 41 (0.283 g, 0.975 mmol) in dry THF (2 mL)under argon was slowly added LDA (1.64 mL, 1.17 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.254 mL, 1.46 mmol) wasadded via syringe. After 15 minutes, compound 63 (0.45 g, 1.46 mmol) wasadded. The resulting mixture was stirred at −78° C. for an additional 4hours. The excess base was quenched with saturated aqueous NH₄Cl (5 mL),and the resulting solution was extracted with EtOAc (3×15 mL). Thecombined organic layers were washed with brine (15 mL), dried overMgSO₄, and filtered, and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography (hexanes/EtOAc,4:1) to afford compound 170 (0.211 g) containing a small amount ofdisubstituted product as a colorless syrup.

Synthesis of Compound 171

Preparation of piperonyl bromide: To a solution of piperonyl alcohol(5.0 g, 32.86 mmol) in anhydrous diethyl ether (80 mL) was added PBr₃(1.56 mL, 16.43 mmol) in one portion, and the resulting mixture wasstirred at room temperature for 3 hours. The mixture was diluted withdiethyl ether (100 mL) and washed with H₂O (2×50 mL), saturated NaHCO₃(2×50 mL), and brine (2×50 mL). The ether layer was dried over anhydrousMgSO₄, and the solvent was removed under reduced pressure to affordpiperonyl bromide (6.43 g, 91%) as a yellow-grey solid.

To a solution of compound 41 (0.154 g, 0.53 mmol) in dry THF (2 mL)under argon was slowly added LDA (0.89 mL, 0.636 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.14 mL, 0.795 mmol) wasadded via syringe. After 15 minutes, piperonyl bromide (0.22 g, 1.02mmol) was added. The resulting mixture was stirred at −78° C. for anadditional 5 hours. The excess base was quenched with saturated aqueousNH₄Cl (5 mL), and the resulting solution was extracted with EtOAc (3×15mL). The combined organic layers were washed with brine (15 mL), driedover MgSO₄, and filtered, and the filtrate was evaporated to dryness.The residue was purified by silica gel column chromatography(hexanes/EtOAc, 4:1) to afford compound 171 (0.101 g, 45%) as acolorless syrup.

Synthesis of Compound 172

Preparation of (3-benzyloxy)benzyl bromide: To a solution of(3-benzyloxy)benzyl alcohol (3.0 g, 14.0 mmol) in anhydrous diethylether (50 mL) was added PBr₃ (0.66 mL, 7.0 mmol) in one portion, and theresulting mixture was stirred at room temperature for 3 hours. Themixture was diluted with diethyl ether (60 mL) and washed with H₂O (2×40mL), saturated NaHCO₃ (2×40 mL), and brine (2×40 mL). The ether layerwas dried over anhydrous MgSO₄, and the solvent was removed underreduced pressure to afford (3-benzyloxy)benzyl bromide (3.76 g, 97%) asa pale yellow solid.

To a solution of compound 41 (0.10 g, 0.344 mmol) in dry THF (2 mL)under argon was slowly added LDA (0.53 mL, 0.379 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.09 mL, 0.517 mmol) wasadded via syringe. After 15 minutes, (3-benzyloxy)benzyl bromide (0.143g, 0.517 mmol) was added. The resulting mixture was stirred at −78° C.for two hours and then allowed to warm up to room temperature over aperiod of two hours. The excess base was quenched with saturated aqueousNH₄Cl (5 mL), and the resulting solution was extracted with EtOAc (3×15mL). The combined organic layers were washed with brine (15 mL), driedover MgSO₄, and filtered, and the filtrate was evaporated to dryness.The residue was purified by silica gel column chromatography(benzene/EtOAc, 95:5) to afford compound 172 (0.054 g, 32%) as acolorless syrup.

Synthesis of Compound 173

Preparation of (4-benzyloxy)benzyl bromide: To a solution of(4-benzyloxy)benzyl alcohol (1.00 g, 4.67 mmol) in anhydrous diethylether (20 mL) was added PBr₃ (0.22 mL, 2.34 mmol) in one portion, andthe resulting mixture was stirred at room temperature for 3 hours. Themixture was diluted with diethyl ether (30 mL) and washed with H₂O (2×20mL), saturated NaHCO₃ (2×20 mL); and brine (2×20 mL). The ether layerwas dried over anhydrous MgSO₄, and the solvent was removed underreduced pressure to afford (4-benzyloxy)benzyl bromide (1.10 g, 85%) asa white solid.

To a solution of compound 41 (0.276 g, 0.95 mmol) in dry THF (2 mL)under argon was slowly added LDA (1.6 mL, 1.14 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.25 mL, 1.425 mmol) wasadded via syringe. After 15 minutes, (4-benzyloxy)benzyl bromide (0.306g, 1.10 mmol) was added. The resulting mixture was stirred at −78° C.for an additional 4 hours. The excess base was quenched with saturatedaqueous NH₄Cl (5 mL), and the resulting solution was extracted withEtOAc (3×15 mL). The combined organic layers were washed with brine (15mL) dried over MgSO₄, and filtered, and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(hexanes/EtOAc, 4:1) to afford compound 173 (0.121 g, 26%) as acolorless syrup.

Synthesis of Compound 174

To a solution of compound 41 (0.10 g, 0.344 mmol) in dry THF (2 mL)under argon was slowly added LDA (0.58 mL, 0.412 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.1 mL, 0.516 mmol) wasadded via syringe. After 15 minutes, 2-nitrobenzyl bromide (0.112 g,0.516 mmol) was added. The resulting mixture was stirred at −78° C. foran additional 4 hours. The excess base was quenched with saturatedaqueous NH₄Cl (5 mL), and the resulting solution was extracted withEtOAc (3×15 mL). The combined organic layers were washed with brine (15mL), dried over MgSO₄, and filtered, and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(benzene:EtOAc, 95:5) to afford compound 174 (0.126 g, 86%) as acolorless syrup.

Synthesis of Compound 175

To a solution of compound 41 (0.10 g, 0.344 mmol) in dry THF (2 mL)under argon was slowly added LDA (0.58 mL, 0.412 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.1 mL, 0.516 mmol) wasadded via syringe. After 15 minutes, 3-nitrobenzyl bromide (0.112 g,0.516 mmol) was added. The resulting mixture was stirred at −78° C. foran additional 4 hours. The excess base was quenched with saturatedaqueous NH₄Cl (5 mL), and the resulting solution was extracted withEtOAc (3×15 mL). The combined organic layers were washed with brine (15mL), dried over MgSO₄, and filtered, and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(benzene/EtOAc, 95:5) to afford compound 175 (0.118 g, 81%) as acolorless syrup.

Synthesis of Compound 176

To a solution of compound 41 (0.10 g, 0.344 mmol) in dry THF (2 mL)under argon was slowly added LDA (0.58 mL, 0.412 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.1 mL, 0.516 mmol) wasadded via syringe. After 15 minutes, 4-methyl-3-nitrobenzyl chloride (96mg, 0.516 mmol) was added. The resulting mixture was stirred at −78° C.for an additional 4 hours. The excess base was quenched with saturatedaqueous NH₄Cl (5 mL), and the resulting solution was extracted withEtOAc (3×15 mL). The combined organic layers were washed with brine (15mL), dried over MgSO₄, and filtered, and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(benzene/EtOAc, 95:5) to afford compound 176 (0.049 g, 32%) as acolorless syrup.

Synthesis of Compound 177

To a solution of compound 41 (0.10 g, 0.344 mmol) in dry THF (2 mL)under argon was slowly added LDA (0.58 mL, 0.412 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.1 mL, 0.516 mmol) wasadded via syringe. After 15 minutes, 4-nitrobenzyl bromide (0.112 g,0.516 mmol) was added. The resulting mixture was stirred at −78° C. foran additional 4 hours. The excess base was quenched with saturatedaqueous NH₄Cl (5 mL), and the resulting solution was extracted withEtOAc (3×15 mL). The combined organic layers were washed with brine (15mL), dried over MgSO₄, and filtered, and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(benzene/EtOAc, 95:5) to afford compound 177 (0.107 g, 73%) as acolorless syrup.

Synthesis of Compound 178

To a solution of compound 41 (0.15 g, 0.516 mmol) in dry THF (3 mL)under argon was slowly added LDA (0.72 mL, 0.62 mmol, freshly preparedfrom n-BuLi and diisopropylamine in THF at −78° C.). The mixture wasstirred at −78° C. for one hour, and then HMPA (0.15 mL, 0.77 mmol) wasadded via syringe. After 15 minutes, 2-methoxy-5-nitrobenzyl bromide(0.19 g, 0.77 mmol) was added. The resulting mixture was stirred at −78°C. for an additional 4 hours. The excess base was quenched withsaturated aqueous NH₄Cl (5 mL), and the resulting solution was extractedwith EtOAc (3×15 mL). The combined organic layers were washed with brine(15 mL), dried over MgSO₄, and filtered, and the filtrate was evaporatedto dryness. The residue was purified by silica gel column chromatography(benzene/EtOAc, 95:5) to afford compound 178 (0.191 g, 81%) as a whitefoam.

Synthesis of Compound 179

A mixture of compound 167 (0.055 g, 0.084 mmol) and 10% Pd/C (55 mg) inHOAc/EtOAc (1:1, 4 mL) was stirred under H₂ (balloon) overnight.Catalyst was removed by filtration and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(hexanes/EtOAc, 3:2) to afford compound 179 (0.033 g, 95%) as a paleyellow syrup.

Synthesis of Compounds 180 and 181

A mixture of compound 170 (0.16 g, −0.31 mmol) containing a small amountof disubstituted product from previous reaction and 10% Pd/C (20 mg) inHOAc/EtOAc (1:1, 6 mL) was stirred under H₂ (balloon) overnight.Catalyst was removed by filtration and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(hexanes/EtOAc, 2:3) to afford compounds 180 (0.084 g) and 181 (0.020 g)as a colorless syrup.

Synthesis of Compound 182

A mixture of compound 172 (0.04 g, 0.082 mmol) and 10% Pd/C (5 mg) inHOAc/EtOAc (1:1, 4 mL) was stirred under H₂ (balloon) overnight.Catalyst was removed by filtration and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(hexanes/EtOAc, 7:3) to afford compound 182 (0.021 g, 65%) as acolorless syrup.

Synthesis of Compound 183

A mixture of compound 173 (0.095 g, 0.195 mmol) and 10% Pd/C (15 mg) inHOAc/EtOAc (1:1, 4 mL) was stirred under H₂ (balloon) overnight.Catalyst was removed by filtration and the filtrate was evaporated todryness. The residue was purified by silica gel column chromatography(hexanes/EtOAc, 3:2) to afford compound 183 (0.069 g, 89%) as acolorless syrup.

Synthesis of Compound 184

A mixture of compound 175 (0.098 g, 0.23 mmol) and 10% Pd/C (15 mg) inEtOAc (3 mL) was stirred under H₂ (balloon) overnight. Catalyst wasremoved by filtration and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography (hexanes/EtOAc,3:7) to afford compound 184 (0.074 g, 81%) as a white solid.

Synthesis of Compound 185

A mixture of compound 177 (0.087 g, 0.205 mmol) and 10% Pd/C (10 mg) inEtOAc (3 mL) was stirred under H₂ (balloon) overnight. Catalyst wasremoved by filtration and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography (hexanes/EtOAc,3:7) to afford compound 185 (0.073 g, 84%) as a pale yellow syrup.

Synthesis of Compound 186

A mixture of compound 178 (0.161 g, 0.353 mmol) and 10% Pd/C (20 mg) inEtOAc (4 mL) was stirred under H₂ (balloon) overnight. Catalyst wasremoved by filtration and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography (hexanes/EtOAc,3:7) to afford compound 186 (0.084 g, 56%) as a white foam.

Synthesis of Compound 187

A mixture of compound 176 (0.043 g, 0.098 mmol) and 10% Pd/C (10 mg) inEtOAc (2 mL) was stirred under H₂ (balloon) overnight. Catalyst wasremoved by filtration and the filtrate was evaporated to dryness. Theresidue was purified by silica gel column chromatography (hexanes/EtOAc,3:7) to afford compound 187 (0.017 g, 42%) as a light red syrup.

The following Scheme 34 illustrates a method of the present inventionthat may be employed to prepare compound 227. More specific detailsregarding this synthesis are provided after the Scheme.

Synthesis of Compound 223 Preparation of(R)-(−)-4-Benzyl-2-oxazolidinone Derivative

The following solutions (Solution 1 and Solution 2) were independentlyprepared. Solution 1: Triethylamine followed by trimethyl acetylchloride were added to a solution of compound 189 in THF at 0° C., andthe mixture was stirred for one hour. Solution 2: n-Butyl lithium wasadded to a solution of (R)-(−)-4-benzyl-2-oxazolidinone in dry THF at−78° C., and the mixture was stirred for one hour.

Solution 2 was added to Solution 1 via cannula. The resultant mixture at0° C. was allowed to warm to room temperature. After stirring at roomtemperature for 24 hours, the mixture was diluted with a saturatedNaHCO₃ solution, and extracted with CH₂Cl₂. The organic layers werecombined, washed with H₂O, dried over MgSO₄, filtered, and the filtrateconcentrated in vacuo. The residue was purified by column chromatographyon silica gel to afford the desired (R)-(−)-4-benzyl-2-oxazolidinonederivative.

n-BuLi was slowly added to a solution of diisopropylamine in dry THF at−78° C. The reaction mixture was stirred at −78° C. under argon for 1hour at −78° C. The (R)-(−)-4-benzyl-2-oxazolidinone derivative in THFat −78° C. was added and the reaction mixture was stirred for 1 hour. Asolution of 3-methylbenzyl bromides in THF was then added in one portionto the reaction mixture. The resulting mixture was warmed to 0° C. andstirred for an additional 2 hours. The excess base was quenched at 0° C.with saturated aqueous NH₄Cl, and the resulting solution was extractedwith CH₂Cl₂. The organic layers were combined, washed successively withsaturated NaHCO₃ and H₂O, dried over MgSO₄, filtered, and the filtratewas concentrated in vacuo. The residue was purified by columnchromatography on silica gel to give compound 223.

Synthesis of Compound 224

Compound 223 and 10% Pd/C in EtOAc was stirred under H₂ (balloon) for 20hours. The mixture was filtered through Celite and the filtrate wasconcentrated in vacuo. The residue was purified by column chromatographyon silica gel to give compound 224.

Synthesis of Compound 225

Diisopropyl azodicarboxylate was added to a suspension of compound 224in admixture with ZnN₆.2Py and Ph₃P in anhydrous toluene. The mixturewas stirred at room temperature for 18 hours. The mixture wasconcentrated in vacuo, and the residue was purified by columnchromatography on silica gel to give compound 225.

Synthesis of Compound 226

LiOH.H₂O and H₂O₂ (30% in H₂O) were added to a solution of compound 225in THF/H₂O (3:1) at 0° C. The reaction mixture was stirred at 0° C. for3 hours. A solution of Na₂SO₃ in water was then added followed by asolution of 0.5 N NaHCO₃. The mixture was stirred for 2 hours, and thenthe THF was evaporated in vacuo. This aqueous solution was diluted with2N HCl to pH=2 and then extracted with EtOAc. The combined organiclayers were dried over MgSO₄, filtered and the filtrate was evaporatedto dryness. The resulting oil was purified using silica gel columnchromatography to give compound 226.

Synthesis of Compound 227

Compound 226 and 10% Pd/C in EtOAc were stirred under H₂ (balloon) for20 hours. The mixture was filtered through Celite and the filtrate wasevaporated to dryness. The residue was purified by column chromatographyon silica gel to give the desired acid compound.

The acid compound was dissolved in toluene and MeOH and treated withpTsOH.H₂O. The solution was heated at reflux for 1.5 hours using aDean-Stark apparatus. The Dean-Stark apparatus was then removed and Et₃Nwas added to the solution, which was heated at reflux for a further 4hours. The solvent was evaporated and the residue was purified by silicagel column chromatography to give the desired compound 227.

The following tables are provided to define compound structures listedin the utility examples. All of the compounds in these tables wereprepared using methodology described herein or methodology described forsimilar compounds as provided herein.

Structure Compound No. R₁ = OMe, R₂ = OH, R₃ = OMe, 30 R₄ = OMe, Q = O

Structure Compound No. R₁ = OMe, R₂ = OH, 129 R₃ = OMe, R₄ = OMe, Q = O

Structure Compound No. R₁ = Ome, R₂ = OH, 130 R₃ = Ome, R₄ = OMe, Q = O

Structure Compound No. R₁ = OMe, R₂ = OH, 12 R₃ = OMe, R₄ = OMe, Q = O

Structure Compound No. R₃ = OC₂H₅, R₄ = OMe 143 R₃ = OCH₂CH₂CH₃, R₄ =OMe 144 R₃ = OCH(CH₃)₂, R₄ = OMe 145 R₃ = OCH₂C(CH₃) = CH₂, R₄ = OMe 146R₃ = (Cyclobutyl)methoxy, R₄ = OMe 147 R₃ = (Cyclohexyl)methoxy, R₄ =OMe 148 R₃ = OCH₂(CH₂)₃CH₃, R₄ = OMe 149 R₃ = OCH₂(CH₂)₄CH₃, R₄ = OMe150 R₃ = OCH₂(CH₂)₅CH₃, R₄ = OMe 151 R₃ = OCH₂(CH₂)₆CH₃, R₄ = OMe 152

Structure Compound No. R₃ = OMe, R₄ = OMe, Q = O 21 R₃ = OcPent, R₄ =OMe, Q = O 41 R₃ = OMe, R₄ = OMe, Q = NH 46 R₃ = OMe, R₄ = OMe, Q = NBn47 R₃ = OMe, R₄ = OMe, Q = NBOC 52 R₃ = OcPent, R₄ = OMe, Q = NH 58 R₃ =OcPent, R₄ = OMe, Q = NBOC 59 R₃ = OBn, R₄ = OMe, Q = O 92 R₃ = OC₂H₅,R₄ = OMe, Q = O 153 R₃ = OCH₂CH₂CH₃, R₄ = OMe, Q = O 154 R₃ = OCH(CH₃)₂,R₄ = OMe, Q = O 155 R₃ = OCH₂C(CH₃) = CH₂, R₄ = OMe, Q = O 156 R₃ =(Cyclobutyl)methoxy, R₄ = OMe, Q = O 157 R₃ = (Cyclohexyl)methoxy, R₄ =OMe, Q = O 158 R₃ = OCH₂(CH₂)₃CH₃, R₄ = OMe, Q = O 159 R₃ =OCH₂(CH₂)₄CH₃, R₄ = OMe, Q = O 160 R₃ = OCH₂(CH₂)₅CH₃, R₄ = OMe, Q = O161 R₃ = OCH₂(CH₂)₆CH₃, R₄ = OMe, Q = O 162

Structure Compound No. R₁ = OMe, R₂ = OH, R₃ = OMe, R₄ = OMe, Q = O 29R₁ = OMe, R₂ = OBn, R₃ = OMe, R₄ = OMe, Q = NBn 49 R₁ = OMe, R₂ = OH, R₃= OMe, R₄ = OMe, Q = NBn 50

Structure Compound No. R₁ = OMe, R₂ = OBn, R₃ = OMe, R₄ = OMe, Q = O 22R₁ = OMe, R₂ = OBn, R₃ = OcPent, R₄ = OMe, Q = O 42 R₁ = OMe, R₂ = OH,R₃ = OcPent, R₄ = OMe, Q = O 43 R₁ = OMe, R₂ = OBn, R₃ = OMe, R₄ = OMe,53 Q = NBOC R₁ = OMe, R₂ = OBn, R₃ = OMe, R₄ = OMe, Q = NH 54 R₁ = OMe,R₂ = OH, R₃ = OMe, R₄ = OMe, Q = NH 55 R₁ = OMe, R₂ = OBn, R₃ = OcPent,R₄ = OMe, 60 Q = NBOC R₁ = OMe, R₂ = OBn, R₃ = OcPent, R₄ = OMe, 61 Q =NH R₁ = OMe, R₂ = OH, R₃ = OcPent, R₄ = OMe, Q = NH 62 R₁ = F, R₂ = F,R₃ = OMe, R₄ = OMe, Q = O 77 R₁ = OBn, R₂ = OBn, R₃ = OMe, R₄ = OMe, Q =O 79 R₁ = OH, R₂ = OH, R₃ = OMe, R₄ = OMe, Q = O 80 R₁ = OH, R₂ = H, R₃= OMe, R₄ = OMe, Q = O 131 R₁ = H, R₂ = OMe, R₃ = OMe, R₄ = OMe, Q = O132 R₁ = H, R₂ = OH, R₃ = OMe, R₄ = OMe, Q = O 133 R₁ = OcPent, R₂ =OMe, R₃OMe, R₄ = OMe, Q = O 134 R₁ = OBn, R₂ = OMe, R₃ = OMe, R₄ = OMe,Q = O 135 R₁ = OMe, R₂ = OcPent, R₃ = OMe, R₄ = OMe, 136 Q = O R₁ = OMe,R₂ = OPr, R₃ = OMe, R₄ = OMe, Q = O 137 R₁ = H, R₂ = OBn, R₃ = OMe, R₄ =OMe, Q = O 138 R₁ = H, R₂ = H, R₃ = OMe, R₄ = OMe, Q = O 139 R₁ = OPr,R₂ = OMe, R₃ = OMe, R₄ = OMe, Q = O 140 R₁ = H, R₂ = F, R₃ = OMe, R₄ =OMe, Q = O 141 R₁ = OBn, R₂ = H, R₃ = OMe, R₄ = OMe, Q = O 142 R₁ = OMe,R₂ = OBn, 163 R₃ = OCH₂(CH₂)₂CH₃, R₄ = OMe, Q = O R₁ = OMe, R₂ = OH, 164R₃ = OCH₂(CH₂)₂CH₃, R₄ = OMe, Q = O R₁ = NO₂, R₂ = OBn, 165 R₃ =OCH₂(CH₂)₂CH₃, R₄ = OMe, Q = O R₁ = NH₂, R₂ = OH, 166 R₃ =OCH₂(CH₂)₂CH₃, R₄ = OMe, Q = O R₁ = OBn, R₂ = OBn, 167 R₃ = OcPent, R₄ =OMe, Q = O R₁ = H, R₂ = H, 168 R₃ = OcPent, R₄ = OMe, Q = O R₁ = CF₃, R₂= H, 169 R₃ = OcPent, R₄ = OMe, Q = O R₁ = OBn, R₂ = OMe 170 R₃ =OcPent, R₄ = OMe, Q = O R₁, R₂ = OCH₂O, 171 R₃ = OcPent, R₄ = OMe, Q = OR₁ = OBn, R₂ = H 172 R₃ = OcPent, R₄ = OMe, Q = O R₁ = H, R₂ = OBn, 173R₃ = OcPent, R₄ = OMe, Q = O R₁ = NO₂, R₂ = H, 175 R₃ = OcPent, R₄ =OMe, Q = O R₁ = NO₂, R₂ = Me, 176 R₃ = OcPent, R₄ = OMe, Q = O R₁ = H,R₂ = NO₂, 177 R₃ = OcPent, R₄ = OMe, Q = O R₁ = NO₂, R₂ = OBn, 93 R₃ =OcPent, R₄ = OMe, Q = O R₁ = OH, R₂ = OH 179 R₃ = OcPent, R₄ = OMe, Q =O R₁ = OH, R₂ = OMe, 180 R₃ = OcPent, R₄ = OMe, Q = O R₁ = NH₂, R₂ = OH,94 R₃ = OcPent, R₄ = OMe, Q = O R₁ = OH, R₂ = H, 182 R₃ = OcPent, R₄ =OMe, Q = O R₁ = H, R₂ = OH, 183 R₃ = OcPent, R₄ = OMe, Q = O R₁ = NH₂,R₂ = H, 184 R₃ = OcPent, R₄ = OMe, Q = O R₁ = H, R₂ = NH₂, 185 R₃ =OcPent, R₄ = OMe, Q = O R₁ = NH₂, R₂ = Me, 187 R₃ = OcPent, R₄ = OMe, Q= O

Structure Compound No. R₁ = NO₂, R₂ = H, 174 R₃ = OcPent, R₄ = OMe, Q =O R₁ = OMe, R₂ = NO₂, 178 R₃ = OcPent, R₄ = OMe, Q = O R₁ = OMe, R₂ =NH₂, 186 R₃ = OcPent, R₄ = OMe, Q = O

Structure Compound No. R₁ = OH, R₂ = OMe, 181 R₃ = OcPent, R₄ = OMe, Q =O

Utility Examples

In vitro and in vivo biological testing showed that the lactone andlactam components of the present invention exhibit an array of potentbiological activities against targets relevant to rheumatoid arthritis,other inflammatory diseases and non-inflammation related diseases asdescribed below.

As used herein, “treating inflammation” refers to both therapy forinflammation, and for the prevention of the development of theinflammatory response. An effective amount of a compound or compositionof the present invention is used to treat inflammation in a warm-bloodedanimal, such as a human. Methods of administering effective amounts ofanti-inflammatory agents are well known in the art and include theadministration of inhalation, oral or parenteral forms. Such dosageforms include, but are not limited to, parenteral solutions, tablets,capsules, sustained release implants and transdermal delivery systems;or inhalation dosage systems employing fry powder inhalers orpressurized multi-dose inhalation devices. Generally, oral or topicaladministration is preferred for the treatment of inflammation. Thedosage amount and frequency are selected to create an effective level ofthe agent without harmful effects. It will generally range from a dosageof about 0.01 to 10 mg/Kg/day where administered orally orintravenously. Also, the dosage range will be typically from about 0.01to 1 mg/Kg/day where administered intranasally or by inhalation.

Administration of compounds or compositions of the present invention maybe carried out in combination with the administration of other agents.For example, it may be desired to co-administer a glucocorticoid for itseffect on arthritis.

Generation of Reactive Oxygen Species by Activated Neutrophils

Neutrophils comprise over 90% of the leukocytic infiltrate in synovialfluid of rheumatoid arthritis (RA) patients and are believed tocontribute to both the acute and chronic phases of this and many otherinflammatory diseases through release of pro-inflammatory mediators,matrix degradative enzymes and toxic oxygen radicals resulting in tissueinjury. One proposed pathogenic mechanism is that the cells are unableto phagocytose large pro-inflammatory substances such as insolubleimmune complexes or damaged endothelium present in the joint.Consequently, neutrophilic granules fuse with the plasma membrane at thesite of activation, rather than internally with phagocyte vacuoles,allowing extracellular release of pro-inflammatory reactive oxygenspecies (ROS) and other toxic substances.

Neutrophil activation can be measured by quantitation of ROS generatedin vitro. Measurement of ROS allows specific quantitation of apro-inflammatory species and is also a general measure of neutrophilactivation. The most sensitive method for measuring the production ofROS by neutrophils is luminol-enhanced chemiluminescence. Compounds andcompositions of the present invention inhibit the generation of ROS. Theassay system used to evaluate the ability of compounds to inhibit ROSgeneration in neutrophils is indicative of anti-inflammatory activitythat may be efficacious in disease states including but not limited torheumatoid arthritis and inflammatory bowel disease.

Freshly isolated primary human neutrophils (5×10⁶ cells/mL) wereincubated with the required concentrations of compound or vehicle for 30minutes at 37° C. in HBSS buffer (pH 7.4) containing Ca²⁺. 100 nMwortmannin was used as a positive control. Aliquots of each sample weretransferred to a microtitre plate to which luminol (1 μM); obtained fromSigma; Catalogue No. A8511 is added. Activation of the neutrophils wasimmediately initiated by addition of (1 μM) fMLP; obtained from Sigma,Catalogue No. F3506. Light output from each well was recorded for 30minutes in a microplate luminometer. Total light output (integral of thetime-course) was determined for each well. Inhibitory activities of testdrugs against neutrophil ROS generation is expressed as percentageactivity relative to a no drug control (100% activation or generation ofROS) containing 0.25% DMSO. Concentration of test compound required toinhibit the generation of ROS to 50% of control values (IC₅₀'s) weredetermined from concentration-response curves by non-linear regressionanalysis. The results are shown in Table 1.

TABLE 1 Inhibition Of Neutrophil Degranulation By Test Compounds AsMeasured By Ros Production COMPOUND IC₅₀ RANGE (μM) NUMBER <1 1-1010-100 >100 12 X 21 X 30 X 29 X 129 X 130 X 179 X 131 X 43 X 168 X 132 X133 X 54 X 61 X 79 X 47 X 154 X 135 X 46 X 169 X 136 X 153 X 42 X 58 X139 X 41 X 155 X 181 X 180 X 77 X 22 X 171 X 62 X 94 X 182 X 99 X 183 X172 X 55 X 80 X 156 X 186 X 175 X 108 X 174 X 185 X 184 X 176 X 155 X157 X

As shown in Table 1, numerous compounds of the present inventiondemonstrate IC₅₀'s in the range of 0.1-1 μM. This result shows thatthese compounds potently block generation of pro-inflammatory reactiveoxygen species by neutrophils in vitro. This result may arise frominhibition of phosphodiesterase and/or chemical scavenging. Thisproperty is predictive of anti-inflammatory activity in vivo due to theestablished role of ROS-mediated tissue injury, e.g., in rheumatoidarthritis, inflammatory bowel disorders, and psoriasis.

Neutrophil Degranulation (Myeloperoxidase Release)

Neutrophilic granulocytes contain several type of organelles known asgranules. These sub-cellular bodies contain a diverse array ofbacteriocidal agents including proteases and other hydrolytic enzymesthat are essential to the normal inflammatory response but contribute toacute tissue injury when neutrophils are chronically and/orinappropriately activated in disease. One of the characteristic granuleenzymes is myeloperoxidase (MPO) which catalyses the conversion ofhydrogen peroxide to hypohalide. MPO is released into the extracellularmilieu on stimulation of degranulation and is a reliable index ofneutrophil activation. Compounds of the present invention inhibited therelease of neutrophil myeloperoxidase from primary human neutrophilsstimulated with 100 μM fMLP. The assay system used to evaluate theability of a compound to inhibit MPO release from neutrophils isindicative of anti-rheumatoid activity as well as other diseases whereinappropriate neutrophil activation is implicated.

Two tubes of human blood (12 mL) were collected into ACD anti-coagulanttubes (Fisher; Catalogue No. 02-684-29). The blood was mixed with 4 mLof 6% dextran in saline. Blood mixture was collected into a 60 ccsyringe. The syringe was tipped upright onto a bench top for 30 min. Theupper serum layer was overlaid on 4 mL Histopaque (Sigma; Catalogue No.1077-1) in a 15 mL centrifuge tube. The tube was centrifuged for 30 minat 2000 rpm. The supernatant was discarded. The pellet was mixed with 3mL cold distilled H₂O, followed by 1 mL 6 N NaCl after 30 seconds. Thetube was centrifuged for 5 min at 1100 rpm. The pellet in each tube wascombined and washed twice with 10 mL (HBSS); Hank's Balance of SaltSolution (Stem Cell Ltd.; Catalogue No. LMC 75). The cells were countedand diluted to 2×10⁶ cells/mL.

Cells (0.3 mL) were pipetted into pre-labelled microcentrifuge tubes.The cells were incubated with 5 μg/mL cytochalasin B, 10 nM PGE₂ and thedesired concentration of test compound or vehicle (0.5% DMSO) for 5 minat 37° C. Wortmannnin or rolipram was used as a positive control. 100 nMfMLP was added into each tube except control. After 30 min incubation,cells were placed on ice and centrifuged at 13,000 rpm for 3 min. 50 μLof supernatant was pipetted into appropriate wells of a 96-well plate(triplicate). 100 μL of substrate was added into each well (0.53 mMo-dianisidin; Sigma, Catalogue No. D 3252, 0.147 mM H₂O₂ in phosphatebuffer pH6.0). The plate was incubated for 30 min at 37° C. Reaction wasterminated by addition of 50 μL 4N H₂SO₄ into each well. To prepare astandard curve, 200 μL of 1, 0.1, 0.01, and 0.001 mg/mL horseradishperoxidase was pipetted into the wells (triplicate). Plate was read byan ELISA reader at 405 nm. The maximum inhibition of myeloperoxidaseroutinely observed for standard compounds with similar mechanisms ofaction to the compounds of the invention (cAMP PDE inhibitor) was about30-35%. Therefore inhibitory potencies from concentration responsestudies are quoted as IC₁₅ values determined from at least threeseparate experiments using triplicate determinations.

As shown in Table 2, compounds of the invention potently inhibited MPOrelease from stimulated human neutrophils with IC₁₅ values ranging from0.1 to 1 μM. These compounds exhibited a maximum inhibition ofneutrophil MPO release of about 40%, consistent with the effects ofknown PDE4 inhibitors such as rolipram (data not shown). This resultshows that these compounds are able to concentration-dependently inhibitthe degranulation response of human neutrophils stimulated with fMLP.Since neutrophil degranulation is considered a major effector of tissueinjury mediated by this cell type in a number of inflammatory diseases(e.g., psoriasis, rheumatoid arthritis), inhibition of this response bythese compounds indicates clinical utility of compounds for these andother related disease states.

TABLE 2 Inhibition Of Neutrophil Degranulation As Measured ByMyeloperoxidase Release COMPOUND IC₁₅ RANGE (μM) NUMBER .1-1 1-10 10-10030 X 21 X 78 X 42 X

Neutrophil Chemotaxis

The process of chemotaxis (directed leukocyte migration up a chemokinegradient) is essential to the accumulation of high numbers ofneutrophils associated with pathological manifestations of inflammation(e.g., deterioration in the rheumatoid joint). Chemotaxis is a primarymechanism whereby neutrophils migrate from the blood vessel lumen to thesite of inflammation and therefore is a common process associated withneutrophil mediated tissue injury in many inflammatory diseases. Withinthe present invention it was discovered that the test compoundsconcentration-dependently inhibited chemotaxis of primary humanneutrophils in response to fMLP. These compounds also inhibitinterleukin 8 (IL-8) mediated chemotaxis (data not shown). Specificinhibition of neutrophil migration to the inflamed joint is a validtherapeutic target in rheumatoid arthritis and many inflammatorydiseases. This in vitro assay system used to evaluate the ability ofcompounds to inhibit chemotaxis is indicative of in vivo anti-rheumatoidactivity and activity against inflammatory diseases where neutrophilsare implicated in the associated tissue injury.

Chemotaxis buffer containing the chemoattractant fMLP (10 μM) was addedto each well of a chemotaxis plate and a filter was inserted ensuringcontact between the filter and the chemotaxis buffer in the well. Asubmaximal concentration of chemoattractant was used which had beendetermined in previous experiments. For determination of spontaneouscell migration certain wells did not receive chemoattractant, butinstead received buffer only. Freshly isolated neutrophils (1×10⁶) wereincubated with vehicle (0.125% DMSO)±test compound for 1 hr at 37° C.Treatment and control cell suspensions were then gently resuspended and20 μL of cells was added to the top side of the filter on each well. Theplate was incubated for 1.5 hours at 37° C. under 5% CO₂. Cells werethen removed from the top side of the filter by aspiration and theentire plate was centrifuged. The filter was removed and knownconcentrations of cells were added to unused wells to prepare a standardcurve. XTT (Sigma; Catalogue No. X 4251)/PMS (Sigma; Catalogue No. P7626) solution prepared in buffer was added to each well and the cellswere further incubated 1-2 hours and measured for absorbance at 450 nm.Absorbance values were converted to cell numbers using the standardcurve. IC₅₀ values are averages from at least three separate experimentswith triplicate determinations.

Table 3 shows the inhibitory potency of compounds of the presentinvention against chemotaxis induced by 10 nM fMLP. Several of thecompounds display IC₅₀'s of less than 10 μM in this assay system. Thephosphodiesterase IV inhibitor Rolipram, used as a positive control,inhibited neutrophil chemotaxis by 85% at 12.5 μM under these assayconditions (data not shown). In control experiments it was found thatthe compounds did not significantly affect general neutrophil motility(in absence of chemoattractant). Thus, the compounds are effective ininhibiting directed migration of human neutrophils in response to abacterially derived peptide, fMLP.

TABLE 3 Inhibition Of Fmlp-Induced Human Neutrophil Chemotaxis COMPOUNDIC₁₅ RANGE (μM) NUMBER .1-1 1-10 10-100 12 X 29 X 30 X 129 X 130 X 21 X

Inhibition of TNF-α Production in Concanavalin-A Stimulated PrimaryHuman CD4+ T-Lymphocytes

Activated T-lymphocytes are known to produce TNF-α and may constitute asignificant source of this important inflammatory mediator in localizedregions of inflammation such as the rheumatoid synovium or psoriaticlesions. Supernatants from conA-stimulated primary human CD4+ T-cellsthat had been incubated in the presence or absence of a test compoundwere analyzed for TNF-α using an ELISA system. (Pharingen; recommendedanti-TNF antibody set 18631-D and 18642-D). Substantial quantities ofTNF-α were induced in vehicle treated T-cells stimulated with conA. Asshown in Table 4, compounds of the present invention were able topotently inhibit the production of T-cell derived TNF-α. This resultcontrasts with the lower potency of these compounds in the inhibition ofLPS-induced TNF-α release in human whole blood. This may be due to thediffering sensitivity of monocyte/macrophages versus T-lymphocytes toelevations in intracellular cAMP with respect to regulatory pathwaysimpacting TNF-α production. This result suggests that compounds of theinvention may be used in the treatment of diseases involving productionof TNF-α.

TABLE 4 Effects Of Test Compounds On Tnf-Alpha Production In Con-AStimulated Human Cd4 + T-Cells TNF-alpha IC₅₀ RANGE (μM) COMPOUND <22.0-20 >20 54 X 55 X 62 X 30 X 136  X 43 X ROLIPRAM X ZARDAVERINE X

Inhibition of Human T-Lymphocyte Helper Function Introduction andRational

Many inflammatory diseases with an autoimmune etiology includingrheumatoid arthritis and psoriasis are characterized by an imbalance inthe T-helper cell function of self-reactive T-lymphocyte subsetsresulting in initiation and maintenance of a pro-inflammatory state.This imbalance is often manifested as excessive expression of a Th1phenotype and/or suppression of a Th2 phenotype. T-cells secreting IL-2and INF-γ are designated as Th1 or type 1 cells. These cells areinvolved through direct cell-mediated immunity by the activation ofmacrophages and cytotoxic cellular pathways. Cells producing IL-4, IL-5and IL-10 are termed Th2 or type 2 cells and regulate the hummoralimmune response. Several compounds of the present invention inhibitedTh1 function more potently than Th2 function in vitro in concanavalin Astimulated primary human CD4+ T-cells. Thus, these compounds may havetherapeutic value in being able to selectively suppress Th1 cellswithout affecting Th2 cells to any significant extent thus resulting incorrection of an imbalance in autoimmune inflammatory diseasecharacterized by elevated Th1 responses.

The assay on the primary human T-cells involves their activation byconcanavalin A (conA); (Sigma, Catalogue No. C 5275), followed by ELISAdetection for cytokines for either profile. In the case of Th1-profiles,IL-2 and INF-γ are used as benchmarks while IL-10 is the indicator for aTh2-profile. Biologically, these indicators are useful in that IL-2 isthe major T-cell mitogen while IL-10 represents a powerful yet selectiveimmunosuppressive agent.

Methods

Approximately 60 cc of whole human blood was collected into ACDanti-coagulant vacutainer tubes. 15 mL Ficoll Paque 1077 was aliquotedinto 6 sterile 50 mL conical tubes. 10 mL of blood was slowly layered ontop of the Ficoll Paque by holding the tube upright and resting thepipette tip at the inside edge of the tube and allowing the blood toslowly run down the side of the tube. Tubes were spun at 1700 rpm for 30minutes at room temperature. The plasma layer above the leukocyte bandwas aspirated off and a pasteur pipette was used to lift off theleukocyte band and transfer it to a sterile 50 mL conical tube. Cellsfrom each leukocyte gradiant tube were transferred to a separate 50 mLconical tube. Sterile PBS pH 7.4 was added to each 50 mL conical tubecontaining cells from leukocyte band to a volume of 50 mL. Tubes werespun at 1100 rpm for 10 minutes. Supernatant was aspirated off and thecells were resuspended in 50 mL PBS pH 7.4. Tubes were re-centrifuged at1100 rpm for 10 minutes. Supernatant was aspirated off and the cellswere resuspended in appropriate medium at a density of 5×10⁷ cells/mL or2×10⁶ cells/mL.

Crude Lymphocyte Preparation: Cells from Leukocyte preparation wereresuspended in BASAL media (AcitCyte™) or RPMI 1640 with 10% FBS+2 mML-glutamine at a density of 1×10⁶ cells/mL.

CD4+ T-cell Preparation: Cells from Leukocyte preparation wereresuspended in sterile PBS pH 7.4 supplemented with 2-6% Fetal BovineSerum (FBS) at a density of 5×10⁷ cells/mL. Cells were then ready forisolation.

CD4+ T-Cell Isolation (StemSep™):

I) Immunomagnetic Labeling:

100 μL of antibody cocktail (Stem Cell Technologies, Catalogue No.14062) was added for each mL of cells and mixed well. Cells+Ab cocktailwas incubated on ice for 30 minutes. After 30 minute incubation, 60 μLof magnetic colloid was added for each mL of cells, and mixed well.Cells+Ab cocktail+magnetic colloid were incubated on ice for 30 minutes.After a final 30 minute incubation cells were ready for magnetic cellseparation.

II) Separation Procedure (Gravity Feed):

Sample was loaded into the top of a column. The stopcock was turned toallow the flow of media down through the column, and the media wascollected into a collection tube. PBS supplemented with 2-6% FBS wasadded to the column until three column volumes had been collected (notincluding the volume of the start sample). Cells were washed, counted,and resuspended in RPMI 1640 with 10% FBS+2 mM L-glutamine at a densityof 1×10⁶ cells/mL.

IL-2, IFN-γ, TNFα and IL-10 Assay Procedure

CD4+ T-cells were isolated as described above. Cells were suspended inRPMI 1640 media (Stem Cell Technologies Inc.; Catalogue No. 36750) with10% FBS+2 mM L-glutamine at a density of 1×10⁶ cells/mL. Cells weremaintained on ice as test compounds were prepared. Test compounds wereprepared in a sterile 96-well assay plate at 50× the final desired testconcentration, all wells contained an equal amount of dimethylsulfoxidevehicle. 500 μL of CD4+ T-cells were added to each well of 24-well assayplate. 10 μL of each test compound working solution was added to theappropriate wells; two wells were left as stimulated and non-stimulatedcontrols. 10 μL of concanavalin A (50× the final concentration) at 10μg/mL was added to each well with the exception of the non-stimulatedcontrol wells. Cells were incubated at 37° C. for 48 hours. Conditionedmedia is then assessed by ELISA for the quantity of IL-2, FN-γ, TNFα andIL-10 present.

Results and Discussion

Tables 5A, 5B and 5C represent a synopsis of data across individualswith respects to the potency of compounds of the invention in theirability to inhibit cytokines that are likely to promote or depress a Th1or Th2 response. In Tables 5A, 5B and 5C, the effect of components ofthe present invention on IL-2, IFN-gamma and IL-10 production inperipheral human CD4+ T-cells stimulated with 10 μg/mL Concanavalin Afor 48 hours is seen. IC₅₀'s are averages from at least three separateexperiments performed in triplicate. CD4+ T-cell isolation, incubationconditions and ELISA detection of lymphokines are discussed in theMethods section above.

Many members of this series of compounds (in particular those thatinhibit both PDE4 and PDE3) elicit a cytokine profile of activatedT-cells that can potentially redress the imbalance of Th1 over Th2 cellsseen in rheumatoid arthritis and other inflammatory diseases such aspsoriasis. Table 5A shows examples of some members of this series thathave demonstrated a selective inhibition of a Th1 profile of the conAstimulated CD4⁺ selected cells; in particular, these compounds arenumbered 136, 54 and 30.

While the absolute potency of these compounds vary depending on theanalog used, the effect is clearly different from that shown by Rolipram(Sigma; Catalogue No. R 6520). Rolipram was seen to inhibit both IL-2and IL-10 synthesis by 48 hrs. In contrast, con-A stimulation of IL-10is not inhibited by several compounds of the invention whereas the samesupernatants show reduced levels of IL-2. The depression of a Th1cytokine profile will necessitate the enhancement of the Th2 cells atthe site of inflammation. The added benefit of inhibiting IL-2production might under the right circumstances render reactive T-cellsanergic (i.e., T-cells that cannot respond to their usual mitogenicstimuli via the TCR in the context of MHC II). Alternatively, it couldalso cause apoptosis of self-reactive T-cells. Therefore, this Th1inhibiting, Th2 sustaining property of compounds of the invention wouldprovide the potential to effect therapeutic improvement in Th1-mediateddiseases such as rheumatoid arthritis, psoriasis and inflammatory boweldisease, amongst other diseases.

TABLE 5A Effects Of Test Compounds On Th1 Profiles In Concanavalin AStimulated Primary Human Cd4 + T-CeIls IL-2 IC₅₀ RANGE (μM) COMPOUND0.02-0.2 0.2-2.0 2.0-20 >20 54 X 55 X 62 X 30 X 136  X 140  X 43 XROLIPRAM X ZARDAVERINE X

TABLE 5B Effects Of Test Compounds On Th1 Profiles In Concanavalin AStimulated Primary Human Cd4 + T-Cells INF-GAMMA IC₅₀ RANGE (μM)COMPOUND 0.02-0.2 0.2-2.0 2.0-20 >20 54 X 55 X 62 X 30 X 136  X 140  X43 X ROLIPRAM X ZARDAVERINE X

TABLE 5C Effects Of Test Compounds On Th2 Profiles In Concanavalin AStimulated Primary Human Cd4 + T-Cells IL-10 IC₅₀ RANGE (μM) COMPOUND0.02-0.2 0.2-2.0 2.0-20 >20 54 X 55 X 62 X 30 X 136  X 140  X 43 XROLIPRAM X ZARDAVERINE X

Oxygen Radical Scavenging

Oxidants and free radicals produced by neutrophils and other cells arebelieved to contribute to the pathogenesis of rheumatoid arthritis andother inflammatory diseases. Consistent with this involvement, compoundscapable of inactivating free radicals (antioxidants) haveanti-inflammatory activities in rheumatoid arthritis and otherinflammatory diseases.

Compounds of the present invention inhibited the formation of freeradicals in a standard in vitro assay used to measure anti-oxidantactivity of biological materials. The basis of the assay (an assay kitfrom RANDOX: Total Anti-Oxidant status) is the ability of antioxidantsin a sample to suppress color formation due to the stable radicalcation, ABTS*⁺. The chromogen ABTS (2,2′-Azino-di-[3-ethylbenzthiazolinesulphonate] (Sigma; Catalogue No. A 1888)) (610 μM) is incubated withsubstrate solution (peroxidase (metmyoglobin) (6.1 μM) and H₂O₂ (250μM)) along with a compound of the invention dissolved in DMSO forexactly 3 minutes at 37° C. Production of the radical cation ABTS*⁺which has a relatively stable blue-green color was measured at 600 nM.Antioxidant activity of 100 μM test compound was determined as describedabove. The positive control used was a potent biological antioxidant,Trolox® (6-hydroxy-2.5.7.8-tetramethylchroman-2-carboxylic acid).Inhibition of color formation (antioxidant activity) by test compoundsis expressed relative to the positive control, Trolox® (100%inhibition). The results are shown in Table 6. Color suppression in thisassay may be due to inhibition of the production of, or quenching of,the ABTS*⁺ radical.

TABLE 6 Anti-Oxidant Activity of Compounds Compound Anti-oxidantactivity range (% of control) Number 1-25 25-50 50-75 75-100 43 X 41 X136  X 54 X 12 X 99 X 94 X 55 X 42 X

The anti-oxidant characteristics of these compounds could contribute toanti-inflammatory activities in vivo and be of therapeutic efficacy ininflammatory diseases involving oxygen radicals. Thus, the anti-oxidantactivity of compounds of the invention could constitute an additionalmechanism of anti-inflammatory action in addition to cAMPphosphodiesterase inhibition.

Resiniferitoxin-Induced Mouse Ear Edema-Acute Inflammation

One of the primary characteristics of inflammation is an increase invascular dilation and permeability leading to the extravasation of, andcollection of fluids in, the interstitium, resulting in redness andswelling. Rheumatoid arthritis in particular is characterized bypronounced edema of affected joints resulting in significant pain andstiffness. The mouse ear inflammation model is a standard in vivo assayfor inflammation that is based on an increase in ear weight which isattributable to edema induced by inflammatory mediators. RTX(resiniferitoxin; Sigma; Catalogue No. R 8756)) is a diterpene isolatedfrom the plant Euphorbia poisonii and is an ultrapotent analog ofcapsaicin. RTX acts by selectively stimulating nociceptive andthermal-sensitive nerve endings in tissue, eliciting neurogenic edema.Compounds of the present invention, whether administered topically,intraperitoneally or orally, inhibited development of edema induced bytopical application of RTX. Edema as induced in this model is inhibitedby PDE4 inhibitors and is thus a useful in vivo system fordifferentiating the efficacy of test compounds that possess comparablein vitro potencies.

Mice (CD1, Charles River Laboratories) were separated into groups(n=5-8) and tagged. Control mice had RTX (0.1 μg/ear) applied topicallyto the inner and outer sides of the left ear and vehicle applied to theright ear as a control. For topical administration,experimental/treatment mice received RTX+test compound solution (50μg/ear) on the left ear and acetone on the right ear. Forintraperitoneal (i.p.) administration, 100 mg/kg test compound dissolvedin 100 μL PEG 200:saline (1:1) was injected followed by a 30 minutewaiting period then the standard 30 minute RTX edema induction. For oral(p.o.) administration, 10 mg/kg test compound in 100 μL PEG-200 wasgiven to animals, then edema induced using 0.1 μg/ear RTX after a 1.5hour waiting period. After edema induction, mice were sacrificed, and astandard disc of ear tissue was removed. Each disk of tissue wasimmediately weighed to the nearest {fraction (1/10)}th of a mg. Datawere analyzed by taking the difference of each left ear from the rightear, calculating the mean+/−SEM. Statistical significance was tested by2-sample t-test on the left/right ear weight differences of the controlgroup vs. the experimental group.

Tables 7, 8 and 9 show the efficacy of compounds of the invention in theRTX-induced mouse ear edema model when administered via topical,intraperitoneal or oral routes of administration. It is apparent fromthese data that the compounds effectively inhibit RTX-mediatedinflammation (edema) in the mouse when delivered through any of thetested routes. Efficacy via topical administration is the greatestfollowed by intraperitoneal then oral gavage. The differences inefficacy between the different routes of administration are notsurprising as g.i. absorption and metabolism processes are important fori.p. and oral delivery of compound.

TABLE 7 Inhibition of Resiniferitoxin-Induced Mouse Ear Edema by TopicalAdministration of Test Compound Compound Number % Inhibition of Edema 1285 29 76 30 98 91 27 92 93 21 32 78 93 99 98 103 94 43 98

TABLE 8 Inhibition of Resiniferitoxin-Induced Mouse Ear Edema byIntraperitoneal Administration of Test Compound Compound Number %Inhibition of Edema 12 61 30 28 99 88 43 72 42 38

TABLE 9 Inhibition of Resiniferitoxin-Induced Mouse Ear Edema by OralAdministration of Test Compound Compound Number % Inhibition of Edema 4345 42 41 62 30

Inhibition of Cyclic Nucleotide Phosphodiesterases Inhibition of cAMPPhosphodiesterase 4

Elevation of cAMP in cells involved in inflammation such as neutrophils,endothelial cells, macrophages, eosinophils, basophils, T-lymphocytesetc. generally leads to the down-regulation of an inflammatory cytokineprofile such as the inhibition of tumor-necrosis factor (TNF-α)expression. Expression of the anti-inflammatory cytokine interleukin-10(IL-10) is positively regulated by cAMP in many cells at a site ofinflammation. Since degradation of cAMP in the cell is effected by cAMPphosphodiesterases (PDEs), specific inhibitors to these enzymes are ofinterest. Such compounds would have the effect of elevatingintracellular cAMP in the cells expressing the PDE isoenzymes theyspecifically inhibit. Although there are at least nine differentfamilies of cyclic nucleotide PDEs, the PDE 4 family is of particularinterest. This is because many of the critical cell types effecting theinflammatory response express predominantly PDE 4 over the other PDEs.PDE 4 inhibitors such as rolipram have been shown to specificallyelevate cAMP in inflammatory cells such as neutrophils and eosinophilsand quench their inflammatory phenotype. A therapeutically-effectivePDE4 inhibitor desirably has minimal side-effects, including inductionof gastric acid secretion, emesis and CNS effects. PDE 4 inhibitorswithout harmful side-effects hold great promise as a new generation ofanti-inflammatory therapeutics for diseases including asthma,inflammatory bowel disease, rheumatoid arthritis, psoriasis andallogeneic transplantation, among others.

Compound 12 was screened for activity against 5 of the major classes ofmammalian cyclic nucleotide phosphodiesterase (termed PDE1 through 5).PDE's 1 through 4 utilize cAMP as substrate while PDE 5 uses cGMP. Thebroad specificity PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX;Sigma; Catalogue No. 17018)) was used as a positive control in allassays. PDE's for the various assays were partially purified from thefollowing cells/tissues; PDE 1 (bovine heart), PDE 2 (human platelets),PDE 3 (human platelets), PDE 4 (human promonocytic U937 cells) and PDE 5(human platelets).

Compound 12 was found to inhibit PDE 1,3 and 4 with IC₅₀'s of 89, 45 and5.9 μM respectively. There were no significant inhibitory effects onPDEs 2 and 5. Thus, compound 12 exhibited significant activity andselectivity toward PDE4, the cAMP phosphodiesterase predominant ininflammatory cells. Compounds of the invention may provide therapeuticutility in autoimmune disease, inflammatory diseases or any diseasewhere elevation of intracellular cAMP in the PDE4 expressinginflammatory cells participating in the disease leads to down-regulationof the inflammatory phenotype.

U937 cytoplasmic extracts were prepared by sonicating U937 cells (ATCC:Catalogue No. CRL-159) in lysis buffer (20 mM Tris Cl, 1 mM EDTA, 5 mMβ-mercaptoethanol, 1 μM pepstatin, 1 μg/mL leupeptin, 1 mM benzamidineand 0.1 mM PMSF). Sonicated cell extracts were then centrifuged at70,000 g for 30 minutes and supernatants removed. Sucrose was added to afinal concentration of 0.25 M, aliquoted and stored at −80° C.

PDE reactions were performed for 30 minutes at 37° C. in 20 μL volumesin 1 μM [³H] cAMP (Amersham website http://www.apbiotech.com), 0.5 U/mL5′ nucleotidase (Sigma), 50 mM Tris Cl, 10 mM MgCl pH 7.5. U937 extractwas added such that less than 10% of substrate was consumed. Testcompound or vehicle was added to the desired concentration. Typically,compounds were tested at six 10-fold dilutions ranging from 100 μM to 1nM. Reactions were performed in duplicate. Reactions were terminated byaddition of 200 μL Dowex 1-8 400 Cl⁻ anion exchange resin in a ratio of1 resin:2 methanol:1 H₂O. Samples were mixed by inversion and thenallowed to settle for 2-3 hours. An aliquot of 65 μL was removed, driedon a Lumaplate (Packard; Catalogue No. 6005165) and counted on a PackardScintillation counter (TopCount™) for 1.5 minutes, to provide the datain Table 10.

TABLE 10 Inhibition of cAMP Phosphodiesterase 4 from Human U937 CellsCompound IC₅₀ range (μM) Number 0.1-1 1-10 12 X 21 X 30 X 29 X 129 X 130X 179 X 131 X 43 X 168 X 132 X 133 X 54 X 61 X 79 X 47 X 154 X 135 X 46X 169 X 136 X 153 X 42 X 58 X 139 X 41 X 155 X 181 X 180 X 77 X 22 X 171X 62 X 94 X 182 X 99 X 183 X 172 X 55 X 80 X 186 X 162 X 175 X 187 X 156X 108 X 174 X 185 X 163 X 166 X 158 X 159 X 155 X 160 X 163 X 161 X 184X 95 X 96 X 114 X 176 X 157 X

Table 10 shows the inhibitory activity of compounds of the inventionagainst PDE4 isolated from a human promonocytic cell line, U937.Utilizing the PDE4 assay conditions described here, typical PDE4inhibitors such as Rolipram and Ro-20-1724 (Calbiochem: Catalogue No.557502) give IC₅₀ values in agreement with those found in the literature(reviewed in Schudt et al., 1996). In addition, use of IBMX (Sigma;Catalogue No. 17018) which inhibits PDEs 1, 3 and 4 does not show anyadditional inhibition (data not shown) again consistent with the findingthat the predominant PDE in U937 cells is PDE 4.

Inhibition of PDE 4 (or more accurately, specific isoforms of PDE 4)with subsequent elevation of intracellular cAMP and protein kinase Aactivation is a therapeutic target in inflammatory or autoimmunediseases where the causal cells or tissues involved predominantlyexpress this PDE isoform. With respect to rheumatoid arthritis, the PDE4 inhibitor rolipram has been shown to be active in animal models of thedisease such as collagen-induced arthritis in the rat (Nyman et al.,Clin. Exp. Immunol. 108(3), 415-419, 1997).

Inhibition of cAMP Phosohodiesterase 3

Compounds of the invention were evaluated for inhibitory activityagainst human platelet PDE3 to ascertain whether the PDE4 inhibition andPDE3 inhibition were separable and also the pharmacophore required foreach. Combined PDE4/3 inhibitors may be especially efficacious astherapeutic agents in diseases where the causative/contributory celltypes express both PDE4 and PDE3, for example T-cells in inflammatorydiseases such as arthritis, inflammatory bowel disease, psoriasis andallogeneic transplantation. In such diseases, combined PDE3/4 inhibitorsmay have advantages over a selective PDE4 inhibitor such as rolipram.

Platelet cell extracts were prepared as described above for the U937cells. The PDE3 assay was performed using platelet cell extract asdescribed above for the PDE 4 assay. Platelets contain PDE 2, 3 and 5.However PDE2 and 5 preferentially utilize cGMP, so in an assay with cAMPas a substrate they are not detected. In addition, under the conditionsused in this assay, rolipram is without effect and the known PDE3inhibitor trequinsin (Calbiochem; Catalogue No.382425) is a potentinhibitor confirming that the assay is specific for PDE3.

Table 11 shows IC₅₀'s for compounds of the invention for the inhibitionof PDE3. The PDE3 and PDE4 activities appear to be separable and thecompounds exhibit a wide range of selectivity for PDE4 vs. PDE3. Somecompounds are specific for PDE4, some compounds are more potent againstPDE4 than PDE3, and some compounds are approximately equipotent againstPDE4 and PDE3. Accordingly, compounds of the invention may be selectedfor their PDE4/3 selectivity to enable maximum potency against differentcell types.

TABLE 11 Inhibition of cAMP phosphodiesterase 3 from human plateletsCom- pound IC₅₀ range (μM) Compound IC₅₀ range (μM) Number 1-1010-100 >100 Number 1-10 10-100 >100 12 62 X 21 X 94 X 30 X 182 X 29 X 99X 129 X 183 X 130 X 172 X 179 X 55 X 131 X 80 X 43 X 186 X 168 X 162 X132 X 175 X 133 X 187 X 54 X 156 X 61 X 108 X 79 X 174 X 47 X 185 X 135X 163 X 46 X 166 X 136 X 158 X 42 X 159 X 58 X 155 X 139 X 160 X 41 X163 X 155 X 161 X 181 X 184 X 180 X 95 X 77 X 96 X 22 X 114 X 171 X 176X 157 X

PDE Isozyme Specificity

We then proceeded to demonstrate that PDE isozyme specificity is acharacteristic of the class of compounds. Test compounds (at 100 μM)were screened for activity against PDE 1,2 and 5, using standardbiochemical methods performed at MDS Panlabs (Bothell, Wash., USA). Thebroad specificity PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) wasused as a positive control in all assays. PDE's for the various assayswere partially purified from the following cells/tissues; PDE 1 (bovineheart), PDE 2 (human platelets) and PDE 5 (human platelets). The resultsare shown in Table 12. For purposes of comparison, the effects on PDE3(human platelets) and PDE4 (human U937 monocytic cell line) describedabove are presented again in Table 12.

TABLE 12 PDE Isozyme Specificity % inhibition by 100 μM test CompoundPDE compound number Isozyme 1-25 25-50 50-75 75-100 12 PDE1 X PDE2 XPDE3 X PDB4 X PDE5 X 43 PDE1 X PDE2 X PDE3 X PDE4 X PDE5 X 41 PDE1 XPDE2 X PDE3 X PDE4 X PDE5 X 136 PDE1 X PDE2 X PDE3 X PDE4 X PDE5 X

Displacement of Rolipram from its High Affinity Binding Site (HARBS) oncAMP Phosphodiesterase 4

There is a need for phosphodiesterase 4 inhibitors that do not haveundesirable side effects including nausea and vomiting. Animal modelshave shown that this activity is highly correlated with a compound'sability to displace [³H]-Rolipram from a high affinity binding site fromcells within the brain and central nervous system (CNS) [Duplantier1996, Barnette 1996]. We have used a High Affinity Rolipram Binding Site(HARBS) displacement assay to predict the emetic potential of a compoundof the present invention. Compounds of the present invention displayed alow affinity for the HARBS conformer of PDE4 suggesting that thesecompounds are not likely to be plagued by mechanism-associatedside-effects associated with first generation PDE4 inhibitors such asrolipram.

Female CD1 mice were sacrificed via the intraperitoneal injection of 100μL euthanol, and the brain tissue homogenized in 5 mL of ice-coldTris-HCl, pH 8.00 supplemented with 1.2 MM MgCl₂, 1 mM benzamidine(Sigma; Catalogue No. B 6506) and 0.1 mM PMSF (Sigma; Catalogue No. P7626). Suspension was centrifuged twice at 30,000×G at 4° C. and thesupernatant discarded. The pellet was resuspended in buffer, andadjusted to a protein concentration of 0.5 mg/mL. Drugs to be testedwere dissolved in DMSO and pipetted in triplicate into a 96 wellmicroplate at concentrations ranging from 1 to 30,000 nM. 10 mL ofmembrane preparation was supplemented with 100 μL of 0.235 μM[³H]-Rolipram in DMSO, and 100 μL dispensed into each well of themicroplate. The plate was incubated at 4° C. for 1 hour. Contents of theplate were aspirated through a Whatman GF/C filterplate, and rinsed with4×200 μL ice-cold buffer. Plate was dried overnight, 30 μL of Microscint20 (Packard; Catalogue No. 6013621) was added to each well, and platewas read in the scintillation counter with a sampling time of 2minutes/well. Values representing non-specific binding (defined bycounts obtained using 20 μM rolipram) were subtracted from all datapoints. Triplicate determinations were performed at each concentration.Results are shown in Table 13. PDE4:HARBS indicates the ratio of theIC₅₀ concentration required to inhibited catalytic activity to theconcentration required to displace 50% of rolipram from the highaffinity binding site.

Under these assay conditions rolipram is able to displace ³H-rolipramfrom a high-affinity binding site in mouse brain with an IC₅₀ of about10 nM (data not shown). Thus, rolipram binds with 20-40 fold greateraffinity to its high affinity site than the concentration required forhalf-maximal inhibition of PDE4 catalytic activity. This preferentialaffinity for HARBS over the catalytic conformer has been correlated withthe negative side effects of first generation PDE4 inhibitors; namelyemesis and CNS effects.

The data shown in Table 13 indicates that the tested compounds are muchless potent at binding to this site than rolipram. For instance,rolipram and compound 43 have very similar IC₅₀'s against the catalyticactivity of PDE4 (280 and 260 nM respectively), however, their HARBSactivities are 10 nM and 250 nM respectively. Thus compound 43 isapproximately 28 times less potent than rolipram for interaction withthe HARBS conformer of PDE4. The ratio of IC₅₀'s for PDE4_(catalytic) toPDE4_(HARBS) for rolipram and compound 43 is 28 and 1.04 respectively.This ratio for compound 43 compares very favorably with values reportedfor second-generation PDE4 inhibitors where HARBS activity has beenreduced through SAR efforts. For example, the ratios reported for SB207499 (Ariflo) and RP 73401 (piclamilast), two specific PDE4 inhibitorsthat have been tested in phase II trials for asthma are 1 and 3respectively. Thus, compounds of the present invention may displayin-vivo emetogenic effects that are much less than rolipram, Ro 20-1724or other first generation PDE4 inhibitors.

TABLE 13 Affinity of Test Compounds for the High Affinity RolipramBinding Site of PDE4 in Mouse Brain 50% displacement of rolipramCompound (μM) Number 0.01-0.1 0.1-1 1-10 >10 PDE4:HARBS 21 X <1 30 X <129 X <1 129 X <1 130 X <1 179 X <1 43 X >1 168 X <1 132 X <1 133 X <1 54X >1 61 X <1 79 X <1 154 X >1 135 X <1 46 X >1 169 X <1 136 X <1 153X >1 42 X <1 58 X >1 41 X >1 155 X <1 181 X <1 180 X >1 77 22 X <1 171 X<1 62 X >1 94 X >1 182 X <1 99 X >1 183 X <1 172 X <1 55 X >1 186 X >1175 X <1 187 X >1 156 X 1 108 X <1 174 X <1 185 X >1 163 X <1 166 X >1158 X <1 159 X >1 155 X <1 160 X <1 163 X <1 161 X <1 184 X >1 176 X >1157 X >1

Potentiation of Forskolin-Induced cAMP Response Element LuciferaseActivity in Human U937 Monocytic Cells

In order to demonstrate the ability of compounds of the presentinvention to elevate cAMP in intact cells, transfection of cells with aplasmid construct containing a cAMP response element (CRE) in a promoterdriving the expression of a luciferase reporter gene (Stratagene; PathDetect™: Catalogue No. 219076) was used to allow sensitive monitoring ofintracellular cAMP levels through detection of light output in aluminometer. Pharmacological treatment of transfected cells with acompound providing a combination of PDE inhibitor and adenylyl cyclaseagonist (receptor or intracellular activator) results in elevatedintracellular cAMP levels detectable from increased light output. cAMPPDE 4 has been shown to be the predominant cyclic nucleotidephosphodieterase activity in U937 cells, and therefore this cell typetransfected with the CRE-luciferase construct can serve as a convenientcellular screening assay for compounds with PDE 4 inhibitory activity.Compounds of the present invention were thereby shown to providepotentiated luciferase expression in U937 cells treated with theadenylyl cyclase activator forskolin.

Human pro-monocytic U937 cells were maintained in RPMI medium containing10% FCS and 2 mM glutamate. U937 cells were transiently transfected asdescribed in Biotechniques Vol. 17(6):1058, 1994. Briefly, cells weregrown in medium containing serum to a density of 5×10⁶ cells/mL and thenresuspended in media containing serum at a density of approximately1×10⁷ cells/mL. 400 μL of cells were transferred into theelectroporation cuvette containing 10 μg of the reporter vector(pCRE-luc) in a volume of 40 μL H₂O. Reporter vector DNA was preparedfrom DH5 α E. coli using the DNA endonuclease free kit (Qiagen) as permanufacturers instructions. U937 cells were electroporated at roomtemperature using a BIORAD electroporator. Capacitance was set to 1050μF and voltage was 280 V. The time constant was noted after eachelectroporation. Cells were then diluted in 4 mL of media and serum and200 μL of cells were plated per well. Cells were allowed to recover for16-18 hours. Cells were then treated with a test compound or vehicle inthe presence or absence of 10 μM forskolin for 4 hours at 37° C.

The luciferase assay was performed as per manufacturers instructions(Tropix). Briefly, cells were centrifuged for 4 minutes at 1200 rpm andmedia supernatant was removed. Cell pellets were lysed in 15 μL Lysisbuffer (Tropix). Luciferase assay was performed using 10 μL of celllysate with 10 μL of buffer A and 25 μL buffer B. Luciferase activitywas obtained using a luminometer with a 5-second delay followed by aread time of 10 seconds.

As shown in Table 14, compounds of the invention potentiate theinduction of luciferase activity in U937 cells treated with 10 μMforskolin. Eleven compounds within the series induce CRE-luciferase atconcentrations between 0.1 and 1 μM. None of the test compounds on theirown induced significant luciferase activity indicating a low basaladenylyl cyclase activity in these cells. This result demonstrates thatthese compounds are capable of elevating cAMP levels in a cell linepredominantly expressing PDE 4 consistent with the observations in theenzymatic assays.

There is a broad correlation between in vitro PDE4 inhibitory activityand CRE luciferase induction potency.

The CRE luciferase assay or variants (different cell types or constructcharacteristics) thereof serves as a convenient cellular SARbackup/validation assay to in-vitro PDE 4 enzymatic assays for efficacyoptimization for compounds of the present invention.

TABLE 14 Potentiation of CRE-Luciferase Activity by Test Compounds inU937 Cells Co-Incubated with the Adenylyl Cyclase Activator ForskolinCom- pound IC₅₀ range (μM) Compound IC₅₀ range (μM) Number 0.1-11-25 >25 Number 0.1-1 1-25 >25 12 X 22 X 21 X 171 X 30 X 62 X 29 X 94 X129 X 182 X 130 X 99 X 179 X 183 X 131 X 172 X 43 X 55 X 168 X 80 X 132X 186 X 133 X 162 X 54 X 175 X 61 X 187 X 79 X 156 X 47 X 108 X 154 X174 X 135 X 185 X 46 X 163 X 169 X 166 X 136 X 158 X 153 X 159 X 42 X155 X 58 X 160 X 139 X 163 X 41 X 161 X 155 X 184 X 181 X 176 X 180 X157 X 77 X

Effects of Compounds of the Invention on Growth of TransformedCells—Potential Anti-cancer Activities Introduction

The BCR-ABL transformed human myeloid leukemia derived cell line, K-562(ATCC; Catalogue No. CRL 243) was used to determine how compounds of thepresent invention affect transformed cell growth. The elevation ofintracellular cAMP is one way to cause cell-cycle arrest or apoptosis ina number of malignancies, in particular certain classes of leukemias(e.g., CLL). Such intracellular mechanisms (i.e., cAMP) have beenreported to bring about differentiation of the un-differentiatedleukemic clones. In particular, it has been shown that elevation ofintracellular cAMP (using a cylic AMP analogue) in p210 BCR-ABLtranformed myleoid leukemia cells is anti-proliferative via inhibitionof cyclin dependent kinase 4 and subsequent down-regulation of c-myc.Thus, the anti-proliferative capacity of compounds of the presentinvention in cultured K-562 cells was compared with several standardphosphodiesterase inhibitors using a 3H-thymidine uptake assay.

Methods

90 μL K562 cells (human chronic myelogenous leukemia cells) were seededinto sterile 96-well assay plates at a density of 1×10⁵ cells/mL in RPMI1640 supplemented with 10% FBS/2 mM L-glutamine. Samples to be testedwere prepared in a sterile 96-well assay plate at 10× the finalconcentration desired. All sample dilutions contained equal amounts ofDMSO to compensate for the % DMSO in the highest sample concentrationused. Nine concentrations of test compound were examined for effects ongrowth up to a maximal concentration of 100 μM. 10 μL of the samples andcontrols (DMSO/normal growth control) were added to the aliquoted cells.The cells were incubated at 37° C./5% CO₂ for 48 hours. Following 48hour incubation, 20 μL of ³H-thymidine was added to each well for afinal concentration of 1 μCi/mL. The cells were then incubated at 37°C./5% CO₂ for 4 to 6 hours. Following 4-6 hour thymidine pulse, plateswere wrapped in plastic and frozen in a −20° C. frost-free freezerovernight. Cells were harvested and ³H-thymidine counts determined. Inorder to distinguish between cytotoxicity and cytostatic activity,growth curves were prepared where the average CPM value of the seedingdensity of cells was plotted on the same curve as the average CPM valuefor the maximum proliferation attained after 48 hours (DMSO growthcontrol cells). The cells were diluted 1:2 for a concentration range of7.8×10³ cells/mL to 2×10⁶ cells/mL. 90 μL of each cell dilution wasseeded in sterile 96-well assay plated and allowed to equilibrate forapproximately 4 hours at 37° C./5% CO₂ prior to ³H-thymidine pulse. Thedata in Table 15 was obtained as described above, and more specifically,K562 cells were seeded into 96-well plates at 1×10⁵ cells/mL in RPMI1640 supplemented with 10% FBS/2 mM L-glutamine. Various concentrationsof test compound or vehicle (DMSO) was added and the cells wereincubated at 37° C./5 CO₂ for 48 hours. The cells were then pulsed at37° C./5% CO₂ for 4 to 6 hours with 1 μCi/mL ³H-thymidine. Radioactivityincorporated into DNA was determined after harvesting onto glass fiberfilters and scintillation counting.

Results and Discussion

The data in Table 15 demonstrates that compounds of the presentinvention are capable of causing cell cycle arrest (e.g., compound 179)and are likely therefore to promote cellular differentiation and/orapoptosis. In contrast, known PDE inhibitors (Rolipram, a PDE-4inhibitor and Zadavarine, a PDE4/3 inhibitor) failed to have any effectupon the proliferative ability of the K-562 cells. While both the testedcompounds of the invention and the known PDE inhibitors in this case alltarget PDE-4, only the inventive compounds show a dramatic effect on thegrowth properties of K-562 cells. This may well indicate a novel classof PDE-4 enzymes targeted by these compounds that are not affected byeither rolipram or zardavarine. The ability of compound 179 to inducecell cycle arrest in the K-562 cell line whereas the canonical PDE4 orPDE4/3 inhibitors rolipram and zardaverine are unable to do so suggestthe inventive compounds may be used in the treatment ofmyeloproliferative and lymphoproliferative disorders such as CML and CLLand potentially other malignancies.

TABLE 15 Anti-Proliferative Activity Of Test Compounds In K562 ChronicMyelogenous Leukemia Cells IC₅₀ range (μM) Compound <50 >50  41 X 179 XROLIPRAM X ZARDAVERINE X

Efficacy in Specific Animal Models of Inflammatory Disease andAutoimmunity

Proof of concept studies in specific disease models were undertaken inanimals to further demonstrate the enzymatic, cellular and generalanti-inflammatory activity of the lactone and lactam compounds of thepresent invention. Literature studies have shown that elevation ofintracellular cAMP through administration of phosphodiesteraseinhibitors, adenylyl cyclase activators, or both, can reduce establisheddisease and/or prevent disease development in various animal models ofinflammatory disease. The efficacy of compounds of the invention wasdemonstrated in animal models of Crohn's disease, rheumatoid arthritisand transplant rejection. With respect to Crohn's disease, anestablished pre-clinical model was used; trinitrobenzenesulfonic-acid(TNBS) induced colitis in the rat. For rheumatoid arthritis,collagen-induced arthritis (CIA) in the mouse was employed. To mimichuman transplant rejection, a murine tail skin allograft transplantationmodel was used.

Inflammatory Bowel Disease (Crohn's Disease)

Inflammatory bowel disease (IBD) is an umbrella term for presentlyincurable, chronic, fluctuating inflammatory diseases of thegastrointestinal tract including Crohn's disease and ulcerative colitis.Symptoms of these disorders include abdominal pain (usually in the lowerright side of the abdomen) and diarrhea with rectal bleeding, weightloss and fever as the condition progresses. The etiology of IBD isunknown, however epidemiological studies suggest an association betweendisease and viral infection (particularly measles) in utero or early inlife. The Crohn's and Colitis Foundation of America (CCFA) estimates 1-2million persons in the US suffer from Crohn's and related IBD's withthose of European descent at greater risk. Incidence rates haveincreased significantly in the 60 years since it (Crohn's) was firstdescribed. In the United States alone, the economic costs of thesediseases are estimated at U.S. $1.8-2.6 billion per year.

A common treatment for IBD consists of oral or intracolonicadministration of 5-aminosalicylic acid (5-ASA), an NSAID derivativewhich is cleaved to ASA (the active drug) in the lower g.i. tract. Othermainstay treatments of IBD include corticosteroids andimmunosuppressants (e.g., 6-mercaptopurine or azathioprine) orcombinations thereof. Recently, an anti-TNF-α therapy was approved forthe treatment of severe Crohn's disease that is resistant toconventional therapies. This therapeutic approach validates theimportance of tumour necrosis factor in IBD. Even with the anti-TNFαapproaches there is much room for improvement in current treatmentmodalities from both the point of view of side effects and efficacy.

Compounds of the present invention were tested in thetrinitrobenzenesulfonic acid (TNBS) induced colitis model in rat (Morriset al., Gastroenterology 96:795-803, 1989; Kim, H.-S. and Berstad, A.,Scandinavian Journal of Gastroenterology 27:529-537, 1992; Ward, Lancetii:903-905, 1977; and Shorter et al., Am. J. Dig. Dis. 17:1024-1032,1972)). Advantages of this particular IBD model include (a) diseasedevelopment in the rat is immune-mediated with Th1 T-cells playing animportant role as is thought to be the case in human disease, (b) singleinstillation of TNBS induces disease of consistent severity andpersistence (c) the model is inexpensive, (d) long duration ofinflammation (up to 8 weeks), (e) a variant of the model in whichcolitis is reactivated mimics the relapsing/remitting nature of thehuman disease, (f) lesions are histopathologically similar to those inhumans (g) clinical pathology mimics the human disease including,necrosis, formation of ulcers, granulocytic infiltration, edema of thebowel, diarrhea and adhesions and (h) many drugs used to treat human IBDare active in the TNBS model.

Compound 43 was evaluated for its ability to attenuate the severity ofcolonic damage and inflammation using the TNBS model. For comparison,separate groups of rats with colitis were treated with 5-aminosalicylicacid, a drug commonly used to treat human inflammatory bowel disease,and NCX-456, a novel derivative of 5-aminosalicylic acid that hasrecently been shown to have markedly enhanced anti-inflammatory activity(Wallace et al., Gastroenterology 117: in press, 1999).

Methods

Colitis was induced by intracolonic instillation of the hapten TNBS (60mg/mL) in 0.5 mL of 50% ethanol. Groups of 8 male, Wistar rats weighing175-225 g received compound 43 (10 mg/kg), 5-aminosalicylic acid at 100mg/kg, NCX-456 (100 mg/kg), or vehicle (1% carboxymethylcellulose)intracolonically 1 hour prior to induction of colitis, 1 h afterinduction of colitis and at 12 h intervals thereafter for one week. Anadditional group of rats received saline intracolonically in place ofTNBS/ethanol and was treated with vehicle at the same times as outlinedabove. Body weights were recorded at the beginning of the study and atdays 2 and 7.

The rats were sacrificed on the 7th day after the induction of colitisand the extent of damage and inflammation was assessed. After the ratswere sacrificed, the distal colon was removed and pinned out on a waxplatform. The presence or absence of diarrhea was noted, as well as thepresence and severity of adhesions between the colon and other organs,and the severity and extent of colonic damage. The order of sacrifice ofthe rats was randomized, and the person scoring the injury was not awareof the treatment the rats had received. After scoring, a sample ofcolonic tissue was excised for measurement of myeloperoxidase activityas an index of granulocyte infiltration (see Wallace et al.,Gastroenterology 117: in press, 1999; and Morris et al.,Gastroenterology 96:795-803, 1989). This tissue sample was 1 cm long(along the axis of the colon) and 5 mm wide and was taken from a regionof macroscopically visible damage (or the corresponding region in anyrats in which there was no damage). The remainder of the tissue wasfixed in neutral buffered formalin and processed by routine methods forsubsequent evaluation by light microscopy. In a blinded manner, thesample of colonic tissue from each rat was examined for evidence ofmucosal ulceration and inflammation. The percentage of the luminalsurface of the section in which ulceration was present was calculated.

Results

One rat in the vehicle-treated group was excluded from analysis becausethe TNBS was rapidly excreted after its installation into the colon(i.e., colitis failed to develop). One vehicle-treated rat died on day 7and one NCX-456-treated rat died on day 6. In each case, perforation ofthe distal colon was observed during necropsy. The various endpoints ofthis study are summarized in Table 16. In vehicle-treated rats,administration of TNBS resulted in extensive ulceration of the distalcolon, diarrhea and adhesions between the colon and other visceraltissues. The bowel wall thickness was more than double that of healthycontrol rats. The global colitis score in the vehicle-treated group was12±1. Colonic myeloperoxidase activity was increased approximately10-fold over the levels in healthy control rats. Histologically, massiveneutrophil infiltration was evident around sites of mucosal ulceration.In the vehicle-treated rats, almost the entire 1 cm segment of tissueexhibited mucosal ulceration extending to the depth of the muscularispropria. Vehicle-treated rats exhibited a significant loss of bodyweight (˜12%) over the one-week period following TNBS administration(Table 16).

TABLE 16 effect of Compound 43 in a tnbs-induced model of inflammatorybowel disease in the rat Adhesion Bowel MPO Score Damage ThicknessGlobal Activity Histology Body Weight Group Mortality (Incidence)Diarrhea Score (mm) Colitis Score (U/mg) Score Change (%) Vehicle 1/71.4 ± 0.2 5/6 6.9 ± 0.6 2.95 ± 0.35 12.04 ± 0.89 63.2 ± 4.9 96.7 ± 2.4−12.4 ± 0.6 (6/6) 5-ASA (100 0/8 1.4 ± 0.3 1/8 6.3 ± 0.8 2.66 ± 0.6910.41 ± 1.58 67.5 ± 8.8 91.4 ± 5.9 −20.2 ± 3.4 mg/kg) (7/8) 43 (10 0/80.5 ± 0.3 3/8 3.4 ± 0.9* 1.67 ± 0.28^(#)  5.86 ± 1.35** 65.1 ± 8.6 50.6± 10.5^(##) +22.6 ± 1.8^(Ψ) mg/kg) (3/8)^(ζ) NCX-456 1/8 0.4 ± 0.3 3/73.7 ± 0.9* 2.02 ± 0.42  6.60 ± 1.67* 57.8 ± 11.6 46.4 ± 4.1^(##) −5.7 ±0.3 (100 mg/kg) (2/7)^(ζ) Healthy 0/8   0 ± 0 0/8   0 ± 0** 1.13 ±0.07^(#) 1.13 ± 0.07^(#) 5.8 ± 0.6**   0 ± 0^(##) 10.7 ± 0.3 controls(0/8)^(ζζζ) ^(ζ)p < 0.05, ^(ζζζ)p < 0.001 versus the vehicle-treatedgroup (Fisher Exact test) *p < 0.05, **p < 0.01 versus thevehicle-treated group (Mann Whitney U test) ^(#)p < 0.05, ^(##)p<0.01versus the vehicle-treated group (ANOVA and Dunnett's MultipleComparison test)^(Ψp < 0.05 versus the vehicle-treated group (ANOVA and Dunnett's Multiple Comparison test))

Treatment with 5-ASA over the course of one week did not significantlyaffect the incidence/severity of adhesions, colonic damage score, bowelwall thickness, myeloperoxidase activity or histological score (Table16). However, 5-ASA did significantly reduce the incidence of diarrhearelative to the vehicle-treated group. Rats treated with 5-ASA exhibitedsimilar loss of body weight (˜20%) as the vehicle-treated group over thecourse of the one-week study (Table 16).

Treatment for one week with the nitric oxide-releasing derivative of5-ASA, NCX-456, resulted in a significant (50%) reduction in the colonicdamage score and a similar reduction in the histological score (Table16). In the latter case, this reflected a reduction in the extent ofulceration of the 1 cm sample that had been fixed and processed forexamination by light microscopy. NCX-456 also significantly reduced theincidence of adhesions relative to the vehicle-treated group. Mean bodyweights of the rats treated with NCX-456 did not differ significantlyfrom those in the vehicle-treated group (Table 16).

Compound 43 significantly reduced the incidence of adhesions (36% ofvehicle), the colonic damage score (49% of vehicle), the thickness ofthe bowel wall (30% of vehicle) and the histological score (52% ofcontrol) resulting in a total reduction in the global colitis score of51% compared to vehicle treated animals. (Table 16). Compound 43 alsoreduced the incidence of diarrhea compared to vehicle treated animalsalthough the effect was not statistically significant. Compound 43 wasthe only one of the various test compounds that prevented the decreasein body weight caused by administration of TNBS (Table 16). Increases intissue myeloperoxidase (MPO) activity, a marker of tissue neutrophilinfiltration in the colon were not prevented by any of the testcompounds including compound 43.

Discussion

These studies demonstrate the effectiveness of compound 43 in the TNBSmodel of colitis in rats. Compound 43 markedly reduced colonic damage,as assessed both macroscopically and histologically, reduced bowel wallthickness, prevented the decrease in body weight normally observedfollowing TNBS administration and reduced the incidence of adhesionsbetween the colon and other visceral organs. 5-ASA, a drug commonly usedfor the treatment of inflammatory bowel disease in humans, was found tobe ineffective in reducing colonic injury, adhesions, body weightchanges and bowel wall thickness. 5-ASA is only effective in about 50%of the trials involving this model. On the other hand, NCX-456, which isa nitric oxide-releasing derivative of 5-ASA (Wallace et al.,Gastroenterology 117: in press, 1999), exhibited actions in the TNBSmodel that were comparable to those of compound 43. However, whileNCX-456 significantly reduced colonic damage and the incidence ofadhesions, in contrast to compound 43, it did not significantly affectthe changes in body weight following TNBS administration, nor did itsignificantly reduce bowel wall thickness. It is important to note thatthe effects of compound 43 reported herein correspond to a dosing levelof 10 mg/kg. 5-ASA and NCX-456 were administered at 100 mg/kg. It hasalso been shown that the therapeutic effects of NCX-456 are lost whenthe dose is reduced to 50 mg/kg.

None of the tested compounds significantly affected colonic tissuemyeloperoxidase activity. MPO is an enzyme found primarily in theazurophilic granules of neutrophils, thereby serving as a biochemicalindex of neutrophil infiltration. The lack of effect of any of thetested compounds on MPO activity, despite significant reductions in theseverity/extent of colonic damage, may have been a consequence of themethod of sampling of tissue. The tissue samples for MPO were taken fromregions of macroscopically visible damage. The histological evaluationrevealed that areas of damage were always associated with massiveneutrophil infiltration. The large concentrations of neutrophils aroundsites of damage may therefore have “masked” any reduction in totalneutrophil influx that occurred in tissues where damage was reduced bytreatment with the test drugs.

The TNBS rat model of gastrointestinal inflammation is an acceptedpre-clinical model for human IBD. The clinical and histopathologicalmanifestations of disease show good similarity to human disease and manydrugs currently used for treatment of IBD in humans have efficacy inthis model. The efficacy of compound 43 in this model implies that thisand other compounds of the invention may be used in therapy of humaninflammatory disease including Crohn's disease and ulcerative colitisamongst others.

Rheumatoid Arthritis Introduction and Rationale

The collagen-induced arthritis (CIA) model in mice is a suitable modelfor evaluating potential drugs active in human rheumatoid arthritis(Trentham, D. E., Arthritis Rheum. 25:911-916, 1982; Brahn, E., Clin.Orthop. 265:42-53, 1991; Holmdahl, R. et al., Arthritis Rheum. 29:106,1986). It shares many of the molecular, cellular and histopathologicalchanges identified as hallmarks of the human disease; these include (a)pronounced proliferation of cells comprising the joint synovialmembrane, (b) formation of an invasive pannus-like tissue, (c)macrophage, granulocyte and lymphocytic infiltration and (d) destructionof bone and cartilage. Like rheumatoid arthritis, animals with CIAexhibit elevated serum levels of immunoglobulin complexes such asrheumatoid factor (RF) and anti-collagen antibodies and inflammatorycytokines in the synovium such as tumour necrosis factor (TNF-α). Inaddition, involvement of MHC class II-restricted T-helper cellactivation/clonal expansion in the synovium has been demonstrated.Radiographs of affected joints often show erosive changes similar tothose seen in human RA and the progressive arthritis often results in anRA-like joint deformity and dysfunction. In addition, many compoundswhich reduce the symptoms of human disease such as anti-TNF biologics,corticosteroids and DMARDS are efficacious in this animal model. Thedevelopment/progression of disease in the CIA model occurs in both animmune (early) and inflammatory phase thus allowing the assessment of awide range of drugs with diverse pharmacological modes of action.

Compound 43 was evaluated for its ability to affect the development orseverity of arthritis in the murine CIA model when administeredintraperitoneally (10 mg/kg, twice daily) in a prophylactic regimeduring developing disease. Effects of this treatment on disease severitywere assessed by qualitative disease scores, quantitative determinationof paw edema and detailed histopathological examination of affectedjoints. Dexamethasone, a powerful corticosteroid, was used as a positivecontrol in the study.

Methods

Male DBA/1J mice (7-8 weeks of age) were immunized through asubcutaneous injection of 0.1 mL of a collagen-adjuvant emulsion (0.1 mgchick type II collagen in complete Freund's adjuvant) at the base of thetail. Mice were then randomly assigned to treatment or control groups inthe following manner: Compound 43 (n=10); 45% 2-hydroxypropylbetacyclodextrin in 0.9% saline vehicle control (n=10); untreated (n=5)and dexamethasone positive control (n=5). After three weeks the animalswere boosted with a second injection of chick type II collagenemulsified at 1.0 mg/mL in incomplete Freund's adjuvant. This secondinjection is required for reproducible induction of disease. In controlanimals, clinical signs of arthritis manifested as erythema and edema ofthe paws and tarsal/metatarsal joints usually appear within 1-2 weeksfollowing the second immunization. Compounds were evaluated for theirability to delay the onset of or reduce the development of arthritis(prophylactic regime). Vehicle, dexamethasone positive control (0.075mg/kg) and compound 43 (10 mg/kg) were administered twice daily (50microliter per injection) by i.p. injection beginning on the day of thesecond collagen injection. The mice continued to receive doses until thelast animal in the vehicle control group reached the seventh day ofhaving established disease. In this particular case, this necessitatedtreatment for 25 days including the day of the booster injection.

The development of clinical arthritis (disease progression) wasmonitored daily after the second collagen injection. All four limbs wereclinically evaluated by a trained observer unfamiliar (blinded) with thetreatment group identity, and scored on a scale of 0-4 for diseaseseverity (redness and swelling) according to the following criteria.

Score Condition 0 Normal 1 Some joints swollen and red, but not all 2All joints swollen 3 Full inflammation of paw 4 Maximum inflammation, nofurther swelling possible

Inflammation was defined as any redness or swelling (enlargement) of anypart of any paw. Established disease was defined as a qualitative scoreof paw inflammation of 2 or greater, that persists for at least 24hours. In addition, paw widths for all four limbs were measured by ablinded observer daily using precision, constant tension calipers.

At the end of the study each animal was euthanized by an overdose ofhalothane anesthesia. Joints both distal to the knee and including theknee were dissected and analyzed by histology. Limb joints were fixed in10% formalin buffer and decalcified in 10% formic acid for 48 hours,then processed for paraffin embedding. Serial sections (5-7 micrometerthick) were stained with haematoxylin and eosin (H & E).Histopathological alterations of the tarsal and metatarsal joints weregraded “blind” by a certified pathologist and a score assigned based ona ranking system.

Results and Discussion

Approximately 14-16 days after administration of the collagen boosterinjection in Incomplete Freund's Adjuvant (IFA) both mice in theuntreated groups and in the vehicle treated groups began displayingovert signs of clinical arthritis. Clinical signs of arthritis includedswelling, redness and disfigurement of the paws. Clinical disease wasrecorded quantitatively (using precision calipers to measure edema ofthe paw) and qualitatively (disease scores assigned based on theseverity of paw inflammation) on a daily basis once signs were evident.As clinical disease progressed (the last 10 days of the study) in theuntreated and vehicle treated groups of mice it became evident that inthose mice treated with 10 mg/kg compound 43 (i.p., bid) the rate ofdisease progression as assessed by paw score was significantly reduced(data not shown).

Mice were sacrificed on the 25^(th) day after the second collageninjection corresponding to approximately the 10^(th) day of establisheddisease. All four paws from each animal in the study were removed, fixedand processed for blinded histopathological examination by an ACVP boardcertified veterinary pathologist. Joint pathology for each foot wasclassified on a scale of 0 to 4, with 0 being normal and 4 the mostseverely affected. Lesion grades were assigned based on the most severelesion(s) present in the paw according to the following scale:

Histopathological Score Description 0 Normal joint 1 Synovialhyperplasia 2 Synovial hyperplasia and leukocytic infiltration withpannus formation 3 Grade 2 with reabsorption of subchondral bone 4 Lossof joint integrity with massive leukocytic infiltration

The individual paw scores were then summed to obtain a total for eachanimal with a maximum possible score for an animal being 16. Groupvalues were obtained by averaging the individual animal scores.

Table 17 shows the average group values with respect to paw edema,clinical arthritis score and joint histopathological score for compound43 treated animals compared to vehicle treated, dex treated anduntreated mice on Day 25 after the collagen boost.

TABLE 17 effect of Compound 43 on clinical and histopathologicalparameters of disease in the murine collagen-induced arthritis model PawEdema Paw Arthritis Histopathological Compound (1/100 in.) Score Score(range) 43 0.052 ± .0051 3.3 ± .64  7.7 (1-15) Vehicle 0.0891 ± .0088 5.1 ± .75 10.8 (3-16) Untreated 0.093 ± .0139 4.4 ± 1.2  9.0 (7-12)dexamethasone −0.0056 ± .0008   0.8 ± .36  5.4 (3-6)

The data in Table 17 show that the vehicle employed in this study,2-hydroxypropyl betacyclodextrin, does not affect the course of diseasedevelopment whether assessed clinically or histopathologically. Thesedata also show that on the final day of the study (day 25), diseaseseverity (whether assessed clinically or histopathologically) wasreduced in the compound 43 treated mice compared to the vehicle group.As shown in the Table, paw edema, arthritis score and histopathologicalscore were reduced by 42%, 35% and 29% respectively in the compound 43treated group compared to the vehicle group. A 2-way ANOVA analysis ofdata from the last 10 days of the study revealed that the arthritisscores of compound 43 treated animals were significantly lower thanthose of the vehicle treated animals (data not shown).

From examination of time courses for edema and arthritis scores it isclear that the effects of compound 43 become manifest more profoundly asdisease progresses. This strongly suggests that compound effects at thisdosage level would be statistically significant in all diseasecategories if the study had been continued for another week.Additionally, these results argue that compound 43 and other compoundsof the invention may be more effective in treatment of existing disease(therapeutic regimen) rather than prophylactically. The modest effectsof compound 43 in amelioration of CIA reported here may indicate thatthe dosage employed is on the cusp of efficacy and that higher doseswould result in greater inhibition. It is also possible thatbioavailability and metabolism parameters are playing important roles inthe results seen.

The reduction in progression of collagen-induced arthritis in mice bycompound 43 reported herein demonstrates that compound 43 may be used inthe treatment of rheumatoid arthritis and other related inflammatorydiseases. These results (in particular the histopathological scores)also indicate that these compounds may be used in the treatment ofdiseases involving perturbations in the bone and cartilage compartmentsof joints including osteoarthritis and osteopenia. The activity ofcompound 43 in this model supports the in-vitro data reported hereinshowing inhibitory effects of this compound and other compounds of theinvention on neutrophil activation, monocyte/macrophage activation andT-cell Th1 responses.

Transplant Rejection In Vitro Testing: CD4 T Cell Activation,Differentiation and Function Methods

AND-TCR transgenic mice (Kaye J. et al., Nature 341:746-749, 1989) wereused to provide a source of naive-antigen specific CD4+T cells. TheAND-T cell antigen receptor recognizes a peptide derived from pigeoncytochrome C (pcc) in the context of the I-E^(k) class II MHC molecule.

To examine the role of a test compound in naive CD4 T cell activationand proliferation, 1×10⁵ AND-lymph node T cells were cultured with 1×10⁶irradiated B10.BR spleen cells in the presence of varying concentrationsof pcc peptide (0-10 μM) in 96 well plates. Proliferation was assessedby ³H thymidine incorporation. All assay conditions were conducted intriplicate. Cell surface activation phenotype of T cells was assessed byflow cytometric analysis.

The differentiation of naive CD4 T cells toward Th1 and Th2 lineages wasperformed as follows: 1×10⁵ AND-lymph node T cells were cultured with10⁷ B10.BR irradiated spleen cells in 2 ml of culture media with thefollowing supplements: for Th1 cell differentiation, 100 U/mL IFNγ, 25U/mL IL2 and 10 ug/mL anti-IL4; for Th2 cell differentiation, 150 U/mLIL4, 25 U/mL IL2 and 10 ug/mL anti-IFNγ. After 3-4 days the wells weresplit 1:4 with the same additions. After 7 days the cells were harvestedand washed 3 times to remove cytokines in the supernatents. 1×10⁵cultured cells were restimulated with 5×10⁵ irradiated B10BR spleencells+5 μM pcc peptide in 250 μl culture media without any addedcytokines. The supernatents were harvested after 40 hrs and assessed forIL2, IL4 and IFNγ by ELISA. Test compounds were added throughout thedifferentiation of the culture.

Cultured Th1 and Th2 cells generated in the absence of test compoundswere tested for proliferation and cytokine activity as described aboveduring antigen stimulation in the presence of compound.

In Vitro Testing: CD8 T Cell Activation, Differentiation and FunctionMethods

2C-TCR transgenic mice (Sha W. C. et al., Nature 335:271-274, 1988) wereused to provide a source of naive antigen specific CD8+T cells. The 2C-Tcell antigen receptor recognizes the 2C peptide derived from themitochondrial alpha-ketoglutarate dehydrogenase enzyme in the context ofthe Db class I MHC molecule.

To test the efficacy of a test compound in naive CD8+ T cell activationand proliferation, single cell suspensions of 2C lymph node (LN) T cellswere isolated from 2C-TCR transgenic mice. 2C-T cells were stimulatedwith irradiated TAP−/−H-2^(d) splenocytes or L^(d) transfected TAP−/−T2cell line in the presence of varying concentrations of 2C peptide (0-10uM). Proliferation was assessed by ³H thymidine incorporation. Cellsurface activation phenotype of T cells was performed by flow cytometricanalysis

Cytotoxic T cells were generated by activation of 2C-T cells withirradiated H-2^(d) splenocytes. Cells were cultured for 7-10 days in thepresence of 25 U/mL IL2. Cytotoxic killer activity of culture cells wastested with a Cr⁵¹ release assay using T2-L^(d) target cells in thepresence of varying concentrations of 2C peptide. The effect of testcompounds on the differentiation of naive CD8 T cells into cytotoxickiller cells was assessed by addition of test compounds during theprimary activation and culture period. The effect of the test compoundsin cytotoxic CD8 T cell activation and effector function was measuredusing the Cr⁵¹ release assay and ³H thymidine incorporation assay in thepresence of the stated concentrations of compound.

Results and Discussion

Testing of compound 43 and 136 in both a one way and two waymixed-lymphocyte-reaction (MLR) demonstrated efficacy for both compounds(approx. 50% or greater inhibition of proliferation at 20 μM). Overallthe best effect was shown for 136 with nearly 70% inhibition usingeither experimental regimen.

Inhibition of CD8+ T-cell proliferation was also demonstrated by thesecompounds with the stronger effect once again coming from 136 (approx.40% inhibition at 5 μM). Both compounds were equipotent (i.e., approx.50% inhibition at 5 μM), in inhibiting the cyto-toxic killer cellfunction of the same CD8+ T-cells.

Naive CD4+ T-cells proliferation was strongly inhibited by both compound43 and 136 (greater than 70% inhibition @5 μM) while CD4+ T cells thatwere already differentiated into a Th1 or Th2 phenotype demonstratedlesser inhibition in the presence of these compounds. Both compoundshowever demonstrate a stronger inhibition of Th1 (approx. 30%) over Th2cells (0 to 8%). This was also reflected somewhat in the extent ofinhibition of Th1 cytokine (IFNγ) versus the Th2 cytokine (IL-4). Thisis likely to result over the long term in the downmodulation/suppression of Th1 cytokines/phenotype in vivo due to theirregulation by Th2 cytokines. This result thus further substantiates ourfinding of a preferential suppression of Th1 cytokine phenotype over aTh2 cytokine phenotype when compounds of the invention (e.g., 136 andcompound 43) are used in gauging the activation of primary human CD4+cells.

The results of the ex vivo studies discussed above indicate: (a)compound 136 and compound 43 preferentially inhibit CD4+ T-cells overCD8+ T-cells; (b) of the CD4+ T-cells, naive T-cells were stronglyinhibited (>=80%), while committed T-cells (Th1/Th2) were not asstrongly impacted; (c) some of the compounds however, notably compound136, showed a preferential inhibition of the Th1 committed populationover the Th2 committed population of T-cells; (d) also while inhibitionof CD8+ T-cells was not profound, inhibition of their functional“killer” activity was observed for those compounds with dual PDE 4/3activity; and (e) the control phosphodiesterase IV inhibitor, rolipram,was uniformly inhibitory to all T-cell populations with little to nodifferential activity.

In Vivo Testing: Murine Tail Skin Allograft Transplantation Model

Three compounds were evaluated in an in vivo transplant model, namelycompounds 54, 41 and 136.

Methods

Tail skin from donor H-2^(b) C57BL/6 mice was transplanted ontorecipient female H-2^(d) BALB/c mice (Lagodzinski, Z. et al., Immunology71:148-150 (1990)). Five mice were included per group. Seven groups ofmice consisting of four test groups treated with test compounds andthree controls which include untreated, vehicle alone and Cyclosporin A(CsA; Sigma; Catalogue No. C 3662)-treated groups were included in thestudy. Test compounds and CsA were administered twice dailyintraperitoneally at a dose of 10 mg/kg beginning at one day prior totransplantation and for 15 days after transplantation, including the dayof transplantation. Mice were monitored and scored daily over 15 dayspost transplantation for graft rejection.

Results and Discussion

Skin allograft rejection is primarily mediated by T lymphocytes withlittle evidence for a major role of antibodies under most circumstances.Skin allograft rejection requires the activation of helper and cytotoxiceffector T cell populations. Graft rejection was assessed by monitoringallograft necrosis. Because tail skin is visibly distinct from thesurrounding trunk skin of the mouse, the course of rejection can beeasily monitored. Fully intact grafts were scored as 100%. Completegraft rejection was defined as >90% graft necrosis. Acute graftrejection generally proceeds via a series of visually obvious eventsbeginning with swelling and erythema of the graft. These events arefollowed by graft desiccation and scab formation over most or all of thegraft, signaling the loss of the viable graft tissue. Scab formation issubsequently followed by shrinkage and scar formation.

All tested compounds of the present invention demonstrated significantenhancement of graft survival when compared to the control (carrieronly; β-cyclodextrin; Sigma; Catalogue No. C 4767) group (Table 18).

TABLE 18 Effect Of Compounds On Graft Rejection In A Murine Tail SkinAllograft Transplantation Model Allograft survival rate # graftssurviving Average graft (% at 9 days beyond 16 days Compound survival(days) post-transplant) p.t. Untreated   8 ± 1   0 0 Vehicle  8.5 ± 1  0 0 Rolipram 11.5 ± 3.7 50 1 Cyclosporin 13.5 ± 3.3 75 2 136 15.4 ± 1.3100 3  41 11.6 ± 4.6 60 1  54 11.5 ± 3.3 75 0

The control group averaged an 8.5 day survival of the skin allograftswhile the groups treated with compounds 54 and 41 averaged 11 to 12 daysurvival. Compound 136 prolonged graft survival for an average of 15.4days and in so doing exceeded the capacity of the positive controlCyclosporin A (13.5 days). Furthermore, the overall quality (% rejectionof individual grafts) of the surviving grafts were similar to, if notbetter than, those obtained using Cyclosporin A.

By comparison to cyclosporin A, the compounds of the invention are alsoamenable for use in all indications where cyclosporin A is used. Thedifferential activities of these compounds as well as their selectivityin cytokines inhibited argues for a mechanism that will not result inimmunosuppression but instead immunomodulation. The ability of thesecompounds to suppress allograft rejection in this model implies thatthey may be of therapeutic utility in diseases such as multiplesclerosis, inflammatory bowel disease, rheumatoid arthritis, psoriasis,organ transplantation and all autoimmune disorders. For example, manydrugs for the treatment of psoriasis are used in organ transplantationor have demonstrated efficacy in this setting. Thus, efficacy ofimmunomodulatory or immunosuppressive drugs in organ transplantationappears predictive of efficacy in psoriasis, boding well for this seriesof compounds as a therapeutic for psoriasis.

All publications, patents, and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually incorporated by reference.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notto be limited by the specific examples provided herein.

What is claimed is:
 1. A compound of the formula

wherein, each of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 15, 16, 17, 18, and 19, as well as the nitrogen at position 1, isindependently substituted at each occurrence with H, —W or —R⁷(W)_(n),wherein W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X,—OH, —NO₂, —SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸,—OCOR⁸, —OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸,—PR⁸R⁸, —POR⁸, —PO₂R⁸, —PO³R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸,—SONR⁸R⁸, —SO₂NH₂, —SO₂NHR⁸ and —SO₂NR⁸R⁸; R⁷ is a C₁-C₃₀ hydrocarbyl,halocarbyl or hydrohalocarbyl group wherein n of the hydrogen or halogenatoms of R⁷ are substituted by an equal number of W groups independentlyselected at each location; R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl orhydrohalocarbyl group; n is selected from 0, 1, 2, 3, 4 and 5; and X isselected from —Br, —Cl, —F, and —I; or a pharmaceutically acceptablesalt or solvate thereof, or as a single stereoisomer or a mixturethereof.
 2. The compound of claim 1 wherein positions 4 and 6 aresubstituted exclusively with hydrogen.
 3. The compound of claim 1wherein position 19 is substituted with —W.
 4. The compound of claim 1wherein position 19 is substituted with —CN, —X, —OH, —NO₂, —SH, or—OR⁸.
 5. The compound of claim 1 wherein one carbon at positions 17 and18 is substituted with hydrogen.
 6. The compound of claim 1 wherein atleast one carbon at positions 17 and 18 is substituted with —W.
 7. Thecompound of claim 1 wherein at least one carbon at positions 17 and 18is substituted with —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X, —OH,—NO₂, —SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸,—OCOR⁸, or —OR⁸.
 8. The compound of claim 1 wherein at least one carbonat positions 17 and 18 is substituted with —CN, —X, —OH, —NO₂, —SH, or—OR⁸.
 9. The compound of claim 1 wherein only one of the carbons atpositions 17, 18 and 19 is substituted with hydrogen.
 10. The compoundof claim 1 wherein the carbon at position 7 is substituted exclusivelywith hydrogen.
 11. The compound of claim 1 wherein the carbon atposition 3 is substituted with hydrogen, R⁸ or X.
 12. The compound ofclaim 1 wherein the carbons at positions 9 and 10 are substituted withhydrogen.
 13. The compound of claim 1 wherein the carbons at positions11, 12, and 13 are independently substituted with hydrogen and —W. 14.The compound of claim 1 wherein only one of the carbons at positions 11and 12 is substituted with hydrogen.
 15. The compound of claim 1 whereina carbon selected from positions 11 and 12 is substituted with —CN, —X,—OH, —NO₂, —SH, or —OR⁸.
 16. The compound of claim 1 wherein at leastone carbon from positions 11, 12, and 13 is substituted with —R⁷(W)_(n).17. The compound of claim 1 wherein at least one carbon from positions11, 12, and 13 is substituted with C₁-C₆hydrocarbyl, C₁-C₆halocarbyl orC₁-C₆hydrohalocarbyl.
 18. The compound of claim 1 wherein exactly two ofthe carbons at positions 11, 12 and 13 are substituted with hydrogen.19. The compound of claim 1 wherein W is selected from, —OCHO, —OH,—OCOR⁸, and —OR⁸.
 20. The compound of claim 1 wherein W is selected from—NH₂, —CN, —X, —OH, —NO₂, —SH, —NHR⁸, —NR⁸R⁸, —OR⁸, and —SR⁸.
 21. Thecompound of claim 1 wherein W is —OR⁸.
 22. The compound of claim 1wherein R⁷ is a C₁-C₁₀ hydrocarbyl group wherein n of the hydrogen orhalogen atoms of R⁷ are substituted by an equal number of W groupsindependently selected at each location.
 23. The compound of claim 1wherein R⁷ is selected from alkyl, alkenyl, alkynyl, aryl, aralkyl,alkylaryl, alkenyl-substituted aryl, aryl-substituted alkenyl,alkynyl-substituted aryl, aryl-substituted alkynyl, biaryl, cycloalkyl,cycloalkenyl, bicycloalkyl, bicycloalkenyl, alkylcycloalkyl,alkenylcycloalkyl, alkynylcycloalkyl, aryl-substituted cycloalkyl,cycloalkyl-substituted aryl, aryl-substituted cycloalkenyl,cycloalkenyl-substituted aryl, aryl-fused cycloalkyl and polycycloalkyl.24. The compound of claim 1 wherein R⁸ is a C₁-C₁₀ hydrocarbyl group.25. The compound of claim 1 wherein R⁸ is a C₁-C₁₀ cyclohydrocarbylgroup.
 26. The compound of claim 1 wherein R⁸ is selected from alkyl,alkenyl, alkynyl, aryl, aralkyl, alkylaryl, alkenyl-substituted aryl,aryl-substituted alkenyl, alkynyl-substituted aryl, aryl-substitutedalkynyl, biaryl, cycloalkyl, cycloalkenyl, bicycloalkyl, bicycloalkenyl,alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, aryl-substitutedcycloalkyl, cycloalkyl-substituted aryl, aryl-substituted cycloalkenyl,cycloalkenyl-substituted aryl, aryl-fused cycloalkyl and polycycloalkyl.27. The compound of claim 1 wherein n is
 0. 28. The compound of claim 1wherein position 1 is substituted with hydrogen.
 29. The compound ofclaim 1 having the S configuration at carbon
 3. 30. The compound ofclaim 1 having the R configuration at carbon
 3. 31. The compound ofclaim 1 having the S configuration at carbon
 5. 32. The compound ofclaim 1 having the R configuration at carbon
 5. 33. The compound ofclaim 1 wherein positions 3, 4, 6, 7, 9 and 10 are substituted withhydrogen, one of positions 11 and 12 is substituted with hydrogen, andone of positions 17 and 18 is substituted with hydrogen.
 34. Thecompound of claim 1 wherein at least one carbon from positions 11, 12,and 13 is substituted with C₁-C₆hydrocarbyl, C₁-C₆halocarbyl orC₁-C₆hydrohalocarbyl, and wherein exactly two of the carbons atpositions 11, 12 and 13 are substituted with hydrogen.
 35. The compoundof claim 1 wherein position 19 is substituted with —CN, —X, —OH, —NO₂,—SH, or —OR⁸; one carbon at positions 17 and 18 is substituted withhydrogen; and at least one carbon at positions 17 and 18 is substitutedwith —CN, —X, —OH, —NO₂, —SH, or —OR⁸.
 36. The compound of claim 1wherein positions 3, 4, 6, 7, 9 and 10 are substituted with hydrogen;exactly two of the carbons at positions 11, 12 and 13 are substitutedwith hydrogen; at least one carbon from positions 11, 12, and 13 issubstituted with C₁-C₆hydrocarbyl, C₁-C₆halocarbyl orC₁-C₆hydrohalocarbyl; one of positions 17 and 18 is substituted withhydrogen; position 19 is substituted with —CN, —X, —OH, —NO₂, —SH, or—OR⁸; and at least one carbon at positions 17 and 18 is substituted with—CN, —X, —OH, —NO₂, —SH, or —OR⁸; and one carbon at positions 17 and 18is substituted with hydrogen.
 37. The compound of claim 1 whereinpositions 1, 3, 4, 6, 7, 9, 10, 11 and 18 are substituted with hydrogen;positions 17 and 19 are substituted with OR⁸ where R⁸ is a C₁-C₁₀hydrocarbyl group; and position 12 is substituted with C₁-C₆hydrocarbyl,C₁-C₆halocarbyl or C₁-C₆hydrohalocarbyl.
 38. The compound of claim 1wherein positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 13, 15, 16, and 18 aresubstituted with hydrogen; position 12 is substituted with C₁hydrocarbyl; position 17 is substituted with —O-cyclopentyl; andposition 19 is substituted with —O-methyl.
 39. The compound of claim 38wherein positions 3 and 5 both have the S stereochemistry.
 40. Thepharmaceutically acceptable salt or solvate of the compound of claim 1.41. A pharmaceutical composition comprising a compound of the formula

wherein, each of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 15, 16, 17, 18, and 19, as well as the nitrogen at position 1, isindependently substituted at each occurrence with H, —W or —R⁷(W)_(n),wherein W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X,—OH, —NO₂, —SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸,—OCOR⁸, —OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸,—PR⁸R⁸, —POR⁸, —PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸,—SONR⁸R⁸, —SO₂NH₂, —SO₂NHR⁸ and —SO₂NR⁸R⁸; R⁷ is a C₁-C₃₀ hydrocarbyl,halocarbyl or hydrohalocarbyl group wherein n of the hydrogen or halogenatoms of R⁷ are substituted by an equal number of W groups independentlyselected at each location; R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl orhydrohalocarbyl group; n is selected from 0, 1, 2, 3, 4 and 5; and X isselected from —Br, —Cl, —F, and —I; or a pharmaceutically acceptablesalt or solvate thereof, or as a single stereoisomer or a mixturethereof; and a pharmaceutically acceptable carrier, diluent, orexcipient.
 42. The composition of claim 41 wherein the compound is acompound wherein positions 1, 3, 4, 5, 6, 7, 9, 10, 11, 13, 15, 16, and18 are substituted with hydrogen; position 12 is substituted with C₁hydrocarbyl; position 17 is substituted with —O-cyclopentyl; andposition 19 is substituted with —O-methyl.
 43. The pharmaceuticalcomposition of claim 41 comprising a pharmaceutically acceptable salt orsolvate of the compound of the formula and a pharmaceutically acceptablecarrier, diluent or excipient.
 44. A method for treating or preventingan inflammatory condition or disease in a patient, comprisingadministering to the patient in need thereof a therapeutically effectiveamount of a compound of the formula

wherein, each of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 15, 16, 17, 18, and 19, as well as the nitrogen at position 1, isindependently substituted at each occurrence with H, —W or —R⁷(W)_(n),wherein W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X,—OH, —NO₂, —SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸,—OCOR⁸, —OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸,—PR⁸R⁸, —POR⁸, —PO₂R⁸, —PO₃R⁸, —SR⁸, —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸,—SONR⁸R⁸, —SO₂NH₂, —SO₂NHR⁸ and —SO₂NR⁸R⁸; R⁷ is a C₁-C₃₀ hydrocarbyl,halocarbyl or hydrohalocarbyl group wherein n of the hydrogen or halogenatoms of R⁷ are substituted by an equal number of W groups independentlyselected at each location; R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl orhydrohalocarbyl group; n is selected from 0, 1, 2, 3, 4 and 5; and X isselected from —Br, —Cl, —F, and —I; or a pharmaceutically acceptablesalt or solvate thereof, or as a single stereoisomer or a mixturethereof; where the amount is effective to treat or prevent theinflammatory condition or disease of the patient.
 45. A method formodulating intracellular cyclic adenosine 5′-monophosphate levels withina patient, comprising administering to a patient in need thereof atherapeutically effective amount of a compound of the formula

wherein, each of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 15, 16, 17, 18, and 19, as well as the nitrogen at position 1, isindependently substituted at each occurrence with H, —W or —R⁷(W)_(n),wherein W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X,—OH, —NO₂, —SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸,—OCOR⁸, —OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸,—PR⁸R⁸, —POR⁸, —PO₂R⁸, —PO₃R⁸, —SR⁸, —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸,—SONR⁸R⁸, —SO₂NH₂, —SO₂NHR⁸ and —SO₂R⁸R⁸; R⁷ is a C₁-C₃₀ hydrocarbyl,halocarbyl or hydrohalocarbyl group wherein n of the hydrogen or halogenatoms of R⁷ are substituted by an equal number of W groups independentlyselected at each location; R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl orhydrohalocarbyl group; n is selected from 0, 1, 2, 3, 4 and 5; and X isselected from —Br, —Cl, —F, and —I; or a pharmaceutically acceptablesalt or solvate thereof, or as a single stereoisomer or a mixturethereof; wherein the amount is effective to modulate the intracellularcyclic adenosine 5′-monophosphate levels of the patient.
 46. A methodfor treating or preventing a disease or condition in a patient, wherethe disease or condition is associated with pathological conditions thatare modulated by inhibiting enzymes associated with secondary cellularmessengers, the method comprising administering to a patient in needthereof a therapeutically effective amount of a compound of the formula

wherein, each of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 15, 16, 17, 18, and 19, as well as the nitrogen at position 1, isindependently substituted at each occurrence with H, —W or —R⁷(W)_(n),wherein W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X,—OH, —NO₂, —SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸,—OCOR⁸, —OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸,—PR⁸R⁸, —POR⁸, —PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸,—SONR⁸R⁸, —SO₂NH₂, —SO₂NHR⁸ and —SO₂R⁸R⁸; R⁷ is a C₁-C₃₀ hydrocarbyl,halocarbyl or hydrohalocarbyl group wherein n of the hydrogen or halogenatoms of R⁷ are substituted by an equal number of W groups independentlyselected at each location; R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl orhydrohalocarbyl group; n is selected from 0, 1, 2, 3, 4 and 5; and X isselected from —Br, —Cl, —F, and —I; or a pharmaceutically acceptablesalt or solvate thereof, or as a single stereoisomer or a mixturethereof; wherein the amount is effective to treat or prevent a diseaseor condition associated with pathological conditions that are modulatedby inhibiting enzymes associated with secondary cellular messengers. 47.A method of treating or preventing transplant rejection in a patient,comprising administering to a patient in need thereof a therapeuticallyeffective amount of a compound of the formula

wherein, each of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 15, 16, 17, 18, and 19, as well as the nitrogen at position 1, isindependently substituted at each occurrence with H, —W or —R⁷(W)_(n),wherein W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X,—OH, —NO₂, —SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸,—OCOR⁸, —OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸,—PR⁸R⁸, —POR⁸, —PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸,—SONR⁸R⁸, —SO₂NH₂, —SO₂NHR⁸ and —SO₂R⁸R⁸; R⁷ is a C₁-C₃₀ hydrocarbyl,halocarbyl or hydrohalocarbyl group wherein n of the hydrogen or halogenatoms of R⁷ are substituted by an equal number of W groups independentlyselected at each location; R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl orhydrohalocarbyl group; n is selected from 0, 1, 2, 3, 4 and 5; and X isselected from —Br, —Cl, —F, and —I; or a pharmaceutically acceptablesalt or solvate thereof, or as a single stereoisomer or a mixturethereof; where the amount is effective to treat or prevent transplantrejection in the patient.
 48. A method of treating or preventinguncontrolled cellular proliferation in a patient, comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a compound of the formula

wherein, each of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 15, 16, 17, 18, and 19, as well as the nitrogen at position 1, isindependently substituted at each occurrence with H, —W or —R⁷(W)_(n),wherein W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X,—OH, —NO₂, —SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸,—OCOR⁸, —OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸,—PR⁸R^(8, —POR) ⁸, —PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸,—SONR⁸R⁸, —SO₂NH₂, —SO₂NHR⁸ and —SO₂NR⁸R⁸; R⁷ is a C₁-C₃₀ hydrocarbyl,halocarbyl or hydrohalocarbyl group wherein n of the hydrogen or halogenatoms of R⁷ are substituted by an equal number of W groups independentlyselected at each location; R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl orhydrohalocarbyl group; n is selected from 0, 1, 2, 3, 4 and 5; and X isselected from —Br, —Cl, —F, and —I; or a pharmaceutically acceptablesalt or solvate thereof, or as a single stereoisomer or a mixturethereof; where the amount is effective to treat or prevent uncontrolledcellular proliferation in the patient.
 49. A method of treating orpreventing conditions associated with the central nervous system (CNS)in a patient, comprising administering to a patient in need thereof atherapeutically effective amount of a compound of the formula

wherein, each of the carbons at positions 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 15, 16, 17, 18, and 19, as well as the nitrogen at position 1, isindependently substituted at each occurrence with H, —W or —R⁷(W)_(n),wherein W is selected from —NH₂, —CONH₂, —COOH, —CN, —CHO, —OCHO, —X,—OH, —NO₂, —SH, —COX, —NHR⁸, —NR⁸R⁸, —CONHR⁸, —CONR⁸R⁸, —COOR⁸, —COR⁸,—OCOR⁸, —OR⁸, —BH₂, —BHR⁸, —BR⁸R⁸, —BO₂H₂, —BO₂R⁸R⁸, —PH₂, —PHR⁸,—PR⁸R⁸, —POR⁸, —PO₂R⁸, —PO₃R⁸, —SR⁸; —SOR⁸, —SO₂R⁸, —SONH₂, —SONHR⁸,—SONR⁸R⁸, —SO₂NH₂, —SO₂NHR⁸ and —SO₂R⁸R⁸; R⁷ is a C₁-C₃₀ hydrocarbyl,halocarbyl or hydrohalocarbyl group wherein n of the hydrogen or halogenatoms of R⁷ are substituted by an equal number of W groups independentlyselected at each location; R⁸ is a C₁-C₃₀ hydrocarbyl, halocarbyl orhydrohalocarbyl group; n is selected from 0, 1, 2, 3, 4 and 5; and X isselected from —Br, —Cl, —F, and —I; or a pharmaceutically acceptablesalt or solvate thereof, or as a single stereoisomer or a mixturethereof; where the amount is effective to treat or prevent conditionsassociated with the central nervous system (CNS) in the patient.
 50. Thecompound of claim 1 of the formula:

as a single stereoisomer or a mixture thereof, or a pharmaceuticallyacceptable salt or solvate thereof.